Purine and Pyrimidine Cdk Inhitbitors and Their use for The Treatment of Autoimmune Diseases

ABSTRACT

The present invention relates to the use of an inhibitor of CDK2 and/or CDK7 and/or CDK9, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating a disease associated with antinuclear antibodies, wherein the inhibitor of CDK2 and/or CDK7 and/or CDK9 or pharmaceutically acceptable salt thereof is administered in an amount sufficient to down-regulate the levels of antinuclear antibodies. A further aspect of the invention relates to a combination comprising an inhibitor of CDK2 and/or CDK7 and/or CDK9, or a pharmaceutically acceptable salt thereof, and methylprednisolone, and its use in the treatment of diseases associated with antinuclear antibodies, such as SLE.

The present invention relates to a method of treating diseasesassociated with antinuclear antibodies. More specifically, but notexclusively, the invention relates to methods of treating autoimmunerheumatic diseases such as human systemic lupus erythematosus (SLE), andpharmaceutical compositions and combinations therefor.

BACKGROUND TO THE INVENTION

The purpose of the immune system is to protect the body from potentiallyharmful substances (antigens) such as microorganisms, toxins, cancercells, and foreign blood or tissues from another person or species.Antigens are destroyed by the immune response, which includes productionof antibodies (molecules that attach to the antigen and make it moresusceptible to destruction) and sensitized lymphocytes (specializedwhite blood cells that recognize and destroy particular antigens).

Immune system disorders occur when the immune response is inappropriate,excessive, or lacking. Autoimmune disorders refers to any diseasecharacterized by abnormal functioning of the immune system that causesthe immune system to produce antibodies against its own tissues. This iscaused by a hypersensitivity reaction where the immune system reacts tosubstances that it normally would ignore, i.e. normal “self” bodytissues.

Normally, the immune system is capable of differentiating “self” from“non-self” tissue. Some immune system cells (lymphocytes) becomesensitized against “self” tissue cells, but these faulty lymphocytes areusually controlled (suppressed) by other lymphocytes. Autoimmunedisorders occur when the normal control process is disrupted, or ifnormal body tissue is altered so that it is no longer recognized as“self.”

Autoimmune disorders typically result in destruction of one or moretypes of body tissues, abnormal growth of an organ, or changes in organfunction. The disorder may affect only one organ or tissue type or mayaffect multiple organs and tissues. Organs and tissues commonly affectedby autoimmune disorders include blood components such as red bloodcells, blood vessels, connective tissues, endocrine glands such as thethyroid or pancreas, organs such as the kidney or liver, muscles,joints, and skin.

Examples of autoimmune or autoimmune-related disorders include:Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type Idiabetes, rheumatoid arthritis, systemic lupus erythematosus,dermatomyositis, Sjogren's syndrome, drug-induced lupus erythematosus,multiple scleroris, myasthenia gravis, Reiter's syndrome and Grave'sdisease.

Symptoms of autoimmune diseases vary widely depending on the type ofdisease. However, autoimmune diseases are often associated withnon-specific symptoms such as fatigue, dizziness, malaise (non-specificfeeling of not being well), fever, and low-grade temperature elevations.

Specific autoimmune disease results in the destruction of an organ ortissue resulting in decreased functioning of an organ or tissue (forexample, the islet cells of the pancreas are destroyed in diabetes),and/or an increase in size of an organ or tissue (for example, thyroidenlargement in Grave's disease). Symptoms vary depending on the specificdisorder and the organ or tissue affected.

Autoimmunity is controlled through balanced suppression of the immunesystem. The aim is to reduce the immune response against normal bodytissue whilst leaving intact the immune response against micro-organismsand abnormal tissues. Clinical treatments for autoimmune diseasestypically involve the use of corticosteroids and immunosuppressants(including cyclophosphamide or azathioprine) which reduce the immuneresponse. However, many of treatments available to date are associatedwith severe adverse side effects.

The present invention seeks to provide alternative therapeutic methodsfor treating autoimmune diseases which ideally are capable of reducingsymptoms and controlling the autoimmune process, whilst maintaining theability to fight disease. More particularly, the invention relates tothe treatment of diseases associated with antinuclear antibodies,especially autoimmune diseases such as human systemic lupuserythematosus. The invention also seeks to provide pharmaceuticalcombinations and compositions suitable for treating such disorders.

STATEMENT OF INVENTION

A first aspect of the invention relates to the use of an inhibitor ofCDK2 and/or CDK7 and/or CDK9, or a pharmaceutically acceptable saltthereof, in the preparation of a medicament for treating a diseaseassociated with antinuclear antibodies, wherein the inhibitor of CDK2and/or CDK7 and/or CDK9, or pharmaceutically acceptable salt thereof isadministered in an amount sufficient to down-regulate the levels ofantinuclear antibodies.

A second aspect of the invention relates to a method of treating adisease associated with antinuclear antibodies in a subject, said methodcomprising administering to the subject an inhibitor of CDK2 and/or CDK7and/or CDK9, or a pharmaceutically acceptable salt thereof, in an amountsufficient to down-regulate the level of antinuclear antibodies.

A third aspect of the invention relates to a method of treating adisease associated with antinuclear antibodies in a subject bydown-regulating the level of antinuclear antibodies in said subject,said method comprising administering an inhibitor of CDK2 and/or CDK7and/or CDK9, or a pharmaceutically acceptable salt thereof, in an amountsufficient to down-regulate the level of antinuclear antibodies.

A fourth aspect of the invention relates to a method of treating adisease associated with antinuclear antibodies in a subject bydown-regulating the level of antinuclear antibodies, said methodcomprising administering an inhibitor of CDK2 and/or CDK7 and/or CDK9,or a pharmaceutically acceptable salt thereof, such that said disease istreated.

A fifth aspect of the invention relates to a method of down-regulatingthe level of antinuclear antibodies in a subject, said method comprisingadministering an inhibitor of CDK2 and/or CDK7 and/or CDK9, or apharmaceutically acceptable salt thereof, to said subject in an amountsufficient to down-regulate the level of antinuclear antibodies.

A sixth aspect of the invention relates to a method of down-regulatingthe level of antinuclear antibodies in a cell, said method comprisingcontacting said cell with an inhibitor of CDK2 and/or CDK7 and/or CDK9,or a pharmaceutically acceptable salt thereof, in an amount sufficientto down-regulate the level of antinuclear antibodies in said cell.

A seventh aspect of the invention relates to a pharmaceuticalcomposition for treating a disease associated with antinuclearantibodies, said composition comprising an inhibitor of CDK2 and/or CDK7and/or CDK9, in an amount sufficient to down-regulate the level ofantinuclear antibodies, admixed with a pharmaceutically acceptablediluent, excipient or carrier.

An eighth aspect of the invention relates to a combination comprising aninhibitor of CDK2 and/or CDK7 and/or CDK9, or a pharmaceuticallyacceptable salt thereof, and methylprednisolone.

A ninth aspect relates to a pharmaceutical composition comprising acombination according to the invention and a pharmaceutically acceptablecarrier, diluent or excipient.

A tenth aspect of the invention relates to a pharmaceutical productcomprising an inhibitor of CDK2 and/or CDK7 and/or CDK9, or apharmaceutically acceptable salt thereof, and methylprednisolone as acombined preparation for simultaneous, sequential or separate use intherapy.

An eleventh aspect of the invention relates to a pharmaceuticalcomposition comprising:

an inhibitor of CDK2 and/or CDK7 and/or CDK9, or a pharmaceuticallyacceptable salt thereof; andmethylprednisolone;admixed with a pharmaceutically acceptable diluent, excipient orcarrier.

A twelfth aspect relates to a combination according to the invention inthe preparation of a medicament for treating a disease associated withantinuclear antibodies.

A thirteenth aspect of the invention relates to the use of an inhibitorof CDK2 and/or CDK7 and/or CDK9, or a pharmaceutically acceptable saltthereof, in the preparation of a medicament for treating a diseaseassociated with antinuclear antibodies, wherein the medicament is foruse in combination with methylprednisolone.

A fourteenth aspect of the invention relates to the use ofmethylprednisolone in the preparation of a medicament for treating adisease associated with antinuclear antibodies, wherein the medicamentis for use in combination with an inhibitor of CDK2 and/or CDK7 and/orCDK9, or a pharmaceutically acceptable salt thereof.

A fifteenth aspect of the invention relates to the use of an inhibitorof CDK2 and/or CDK7 and/or CDK9, or a pharmaceutically acceptable saltthereof, and methylprednisolone, in the preparation of a medicament fortreating a disease associated with antinuclear antibodies.

A sixteenth aspect of the invention relates to the use of an inhibitorof CDK2 and/or CDK7 and/or CDK9, or a pharmaceutically acceptable saltthereof, in the preparation of a medicament for the treatment of adisease associated with antinuclear antibodies, wherein said treatmentcomprises administering to a subject simultaneously, sequentially orseparately an inhibitor of CDK2 and/or CDK7 and/or CDK9, or apharmaceutically acceptable salt thereof, and methylprednisolone.

A seventeenth aspect of the invention relates to a method of treating adisease associated with antinuclear antibodies in a subject, said methodcomprising administering to the subject a therapeutically acceptableamount of:

an inhibitor of CDK2 and/or CDK7 and/or CDK9, or a pharmaceuticallyacceptable salt thereof; andmethylprednisolone.

For ease of reference, these and further aspects of the presentinvention are now discussed under appropriate section headings. However,the teachings under each section are not necessarily limited to eachparticular section.

DETAILED DESCRIPTION Inhibitors of CDK2 and/or CDK7 and/or CDK9

The present invention relates to the use of one or more inhibitors ofCDK2 and/or CDK7 and/or CDK9. For the avoidance of doubt, the inhibitormay be an inhibitor of any one of CDK2, CDK7 or CDK9, or any combinationthereof.

In one preferred embodiment, the inhibitor is an inhibitor of CDK2.

In another preferred embodiment, the inhibitor is an inhibitor of CDK7.

In another preferred embodiment, the inhibitor is an inhibitor of CDK9.

In another preferred embodiment, the inhibitor is an inhibitor of CDK7and CDK9.

Methods for assaying CDK activity will be familiar to the skilledartisan. Further details are outlined in the accompanying Examplessection.

Preferably, the inhibitor exhibits an IC₅₀ value for one or more ofCDK2, CDK7 or CDK9 of less than 1 mM, more preferably less than 100 μM,more preferably still, less than 50 μM, more preferably less than 25 μM,more preferably less than 10 μM, even more preferably less than 1 μM or0.5 or 0.1 μM.

Suitable examples of inhibitors of CDK2 and/or CDK7 and/or CDK9 includepurine derivatives, such as those disclosed in EP 0874847B (CNRS), WO03/002565 (Cyclacel Limited), WO 04/016613 (Cyclacel Limited) and WO04/016612 (Cyclacel Limited); and pyrimidine derivatives such as thosedisclosed in WO01/72745, WO 02/079193, WO 03/029248, WO04/043953 (all inthe name of Cyclacel Limited).

In one preferred embodiment of the invention, the inhibitor of CDK2and/or CDK7 and/or CDK9 is selected from the following:

and pharmaceutically acceptable salts thereof.

In one particularly preferred embodiment, the inhibitor of CDK2 and/orCDK7 and/or CDK9 is selected from compounds [1]-[3], [5]-[8], [11] and[12].

In another particularly preferred embodiment, the inhibitor of CDK2and/or CDK7 and/or CDK9 is selected from compounds [4], [9] and [10].

In yet another particularly preferred embodiment, the inhibitor of CDK2and/or CDK7 and/or CDK9 is compound [7].

To date, there has been no suggestion that inhibitors of CDK2 and/orCDK7 and/or CDK9 would be effective in reducing the level of antinuclearantibodies. Nor has there been any teaching or suggestion in the artthat such inhibitors could be used in combination therapy withmethylprednisolone in the treatment of autoimmune disorders.

In one preferred embodiment, the inhibitor of CDK2 and/or CDK7 and/orCDK9 is selected from roscovitine, olomoucine and purvalanol A.

In an even more preferred embodiment of the invention, the inhibitor ofCDK2 and/or CDK7 and/or CDK9 is roscovitine.

Roscovitine or2-[(1-ethyl-2-hydroxyethyl)amino]-6-benzylamine-9-isopropylpurine, isalso described as2-(1-D,L-hydroxymethylpropylamino)-6-benzylamine-9-isopropyl-purine. Asused herein, the term “roscovitine” encompasses the resolved R and Senantiomers, mixtures thereof, and the racemate thereof.

As used herein, the term “CYC202” refers to the R enantiomer ofroscovitine, namely,2-(1-R-hydroxymethylpropylamino)-6-benzylamino-9-isopropylpurine, thestructure of which is shown below.

The in vitro activity of roscovitine is as follows:

Kinase IC₅₀ (μM) Cdk1/cyclin B 2.7 Cdk2/cyclin A 0.7 Cdk2/cyclin E 0.1Cdk7/cyclin H 0.5 Cdk9/cyclin T1 0.8 Cdk4/cyclin D1 14.2 ERK-2 1.2PKA >50 PKC >50

Although the use of roscovitine in the treatment of autoimmune disordersis documented in the art, to date, there has been no suggestion that itwould be effective in reducing the level of antinuclear antibodies. Norhas there been any teaching or suggestion in the art that roscovitinecould be used in combination therapy with methylprednisolone in thetreatment of autoimmune disorders.

For all embodiments of the invention, preferably the roscovitine is inthe form of the R enantiomer, namely2-(1-R-hydroxymethylpropylamino)-6-benzylamino-9-isopropyl-purine,hereinafter referred to as “CYC202”.

Therapeutic Activity

As mentioned above, the present invention relates to the use of aninhibitor of CDK2 and/or CDK7 and/or CDK9, or a pharmaceuticallyacceptable salt thereof, in the preparation of a medicament for treatinga disease associated with antinuclear antibodies, wherein the inhibitorof CDK2 and/or CDK7 and/or CDK9, or pharmaceutically acceptable saltthereof, is administered in an amount sufficient to down-regulate thelevels of antinuclear antibodies.

Antinuclear antibodies (ANAs) are unusual antibodies, detectable in theblood, that have the capability of binding to certain structures withinthe nucleus of the cells. ANAs are found in patients whose immune systemmay be predisposed to cause inflammation against their own body tissues.Antibodies that are directed against one's own tissues are referred toas auto-antibodies. The propensity for the immune system to work againstits own body is referred to as autoimmunity.

The presence of antinuclear antibodies is a hallmark of autoimmunediseases. Antinuclear antibodies are a diverse group of antibodies,often directed to large cellular complexes containing protein andnucleic acid components. The most frequently occurring antinuclearantibodies react with components of DNA-protein or RNA-protein complexes[Van Venrooij W. J. et al; Von Mulen C. A. et al]. Studies haveindicated that the production of these autoantibodies, which aregenerally high-titre, high affinity IgG antibodies is T-cell dependentand driven by the host autoantigen [Reichlin M. et al].

For a disease to be defined as autoimmune, the tissue damage must beshown to be caused by an adaptive immune response to self antigens.Autoimmune diseases can be mediated by autoantibodies and/or byauto-reactive T cells, and tissue damage can result from direct attackon the cells bearing the antigen, from immune-complex formation, or fromlocal inflammation. T cells can be involved directly in inflammation orcellular destruction, and they are also required to sustain autoantibodyresponses. Similarly, B cells may be important antigen-presenting cellsfor sustaining autoantigen-specific T-cell responses.

CD4 T cells selectively activate those B cells that bind epitopes thatare physically linked to the peptide recognized by the T cell. Thus,both autoreactive B cells and autoreactive T cells are required for adisease involving autoimmunity.

Anti-nuclear antibody production can only be studied in vivo since theproduction of these antibodies requires a dysfunctional immune systemcomprising both B and T cells, and a failure to select and destroyimmune cells that recognise self.

In vitro assays for T cell function are appropriate screening tools toidentify compounds that may have the ability to modulate the immuneresponse in the complex situation of autoimmune disease. One such assayis a T cell proliferation assay, further details of which are set forthin the accompanying Examples. In autoimmune diseases involvinganti-nuclear antibody production normal T cell function is required tostimulate autoantibody production by B cells. Accordingly, compoundsaffecting T cell function (one measure of T cell function is the abilityto proliferate in response to an immune stimulus) should also preventthe formation of autoantibodies, by controlling the ability of T cellsto communicate with B cells and in addition by preventing T cells frommigrating to the site of auto-immune damage.

As used herein, the term “antinuclear antibodies” includes both anti-DNAantibodies, anti-RNA antibodies and antibodies directed against proteinnuclear components. Diseases associated with antinuclear antibodiesinclude autoimmune rheumatic diseases and organ specific autoimmunity.

Preferably, the autoimmune rheumatic disease is selected from druginduced lupus, systemic lupus erythematosis (SLE), and rheumatoidarthritis.

Rheumatoid arthritis is a chronic autoimmune disease that involvesinflammation in the lining of the joints and/or other internal organs.Rheumatoid arthritis is a systemic disease that affects the entire bodyand is one of the most common forms of arthritis. It is characterized bythe inflammation of the membrane lining the joint, which causes pain,stiffness, warmth, redness and swelling. The inflamed joint lining, thesynovium, can invade and damage bone and cartilage. Inflammatory cellsrelease enzymes that may digest bone and cartilage. The involved jointcan lose its shape and alignment, resulting in pain and loss ofmovement. Symptoms typically include inflammation of joints, swelling,difficulty moving and pain. Other symptoms include loss of appetite,fever, loss of energy and anemia.

To date, treatment methods focus on relieving pain, reducinginflammation, stopping or slowing joint damage, and improving patientfunction and well-being. Medications can be divided into two groups: (i)symptomatic medications, (such as NSAIDS, aspirin, analgesics andcorticosteroids) which help reduce joint pain, stiffness and swelling;and (ii) disease-modifying antirheumatic drugs, such as low doses ofmethotrexate, leflunomide, D-Penicillamine, sulfasalazine, gold therapy,minocycline, azathioprine, hydroxychloroquine (and other antimalarials),cyclosporine and biologic agents.

More preferably, the autoimmune rheumatic disease is systemic lupuserythematosis (SLE).

Systemic Lupus Erythematosus

In one preferred embodiment, the invention relates to the treatment ofsystemic lupus erythematosus (also known as disseminated lupuserythematosus; SLE; lupus; lupus erythematosus) is a chronic,inflammatory autoimmune disorder that may affect many organ systemsincluding the skin, joints and internal organs. Although people with thedisease may have many different symptoms, some of the most common onesinclude extreme fatigue, painful or swollen joints (arthritis),unexplained fever, skin rashes, and kidney problems. At present there isno cure for SLE.

The disease affects nine times as many women as men and appears morecommon in people of African descent. It may occur at any age, butappears most often in people between the ages of 10 and 50 years. SLEmay also be caused by certain drugs. When this occurs, it is known asdrug-induced lupus erythematosus and it is usually reversible when themedication is stopped.

The course of the disease may vary from a mild episodic illness to asevere fatal disease. Symptoms also vary widely in a particularindividual over time and are characterized by periods of remission andexacerbation. Some of the most common symptoms of lupus include painfulor swollen joints (arthritis), unexplained fever, and extreme fatigue.Other symptoms of lupus include chest pain, hair loss, anemia (adecrease in red blood cells), mouth ulcers, and pale or purple fingersand toes from cold and stress. Some people also experience headaches,dizziness, depression, confusion, or seizures. New symptoms may continueto appear years after the initial diagnosis, and different symptoms canoccur at different times. At its onset, only one organ system may beinvolved. Additional organs may become involved later.

Lupus is characterized by periods of illness, called flares, and periodsof wellness, or remission. Typically, once SLE has been diagnosed, thedoctor will develop a treatment plan based on the patient's age, sex,health, symptoms, and lifestyle. In developing a treatment plan, thedoctor has several goals: to prevent flares, to treat them when they dooccur, and to minimize organ damage and complications.

To date, a number of different treatments are available for SLEsufferers. For people with joint or chest pain or fever, drugs thatdecrease inflammation, called nonsteroidal anti-inflammatory drugs(NSAIDs), are often used. Common side effects of NSAIDs can includestomach upset, heartburn, diarrhea, and fluid retention. Some sufferersalso develop liver, kidney, or even neurological complications.

Antimalarials are another type of drug commonly used to treat lupus.Although they are generally used to treat fatigue, joint pain, skinrashes, and inflammation of the lungs, clinical studies have found thatcontinuous treatment with antimalarials may prevent flares fromrecurring. Side effects of anti-malarials can include stomach upset and,extremely rarely, damage to the retina of the eye.

The mainstay of lupus treatment involves the use of corticosteroidhormones. Corticosteroids work by rapidly suppressing inflammation andcan be given by mouth, in creams applied to the skin, or by injection.Short-term side effects of corticosteroids include swelling, increasedappetite, and weight gain. These side effects generally stop when thedrug is stopped. However, it can often be dangerous to stop takingcorticosteroids suddenly, so there is a need to wean the patient offthem over an extended period of time. Long-term side effects ofcorticosteroids can include stretch marks on the skin, weakened ordamaged bones (osteoporosis and osteonecrosis), high blood pressure,damage to the arteries, high blood sugar (diabetes), infections, andcataracts. Typically, the higher the dose and the longer they are taken,the greater the risk and severity of side effects.

For patients whose kidneys or central nervous systems are affected bylupus, immunosuppressives may be used. Immunosuppressives restrain theoveractive immune system by blocking the production of immune cells.Side effects may include nausea, vomiting, hair loss, bladder problems,decreased fertility, and increased risk of cancer and infection. Therisk for side effects increases with the length of treatment. As withother treatments for lupus, there is a risk of relapse after theimmunosuppressives have been stopped.

The present invention provides alternative therapeutic treatments forSLE and other autoimmune rheumatic diseases. In particular, theinvention aims to provide alternative treatments to immunosuppressantsand steroids, which at least in the acute phase of the disease haveexcellent therapeutic effects but which lack major long term sideeffects.

In Vivo Studies

Studies were undertake to investigate the efficacy of CYC202 andcombinations of CYC202/methylprednisolone in mice.

NZB/W F1 hybrid mice spontaneously develop a severe autoimmune diseasereminiscent of human systemic lupus erythematosus. The disease manifestswith early antinuclear antibody formation, development of an immunecomplex glomerulonephritis with proteinuria and progression to renalinsufficiency with time, that also causes premature mortality in thesemice. It is a disease of T and B cell dysfunction with the formation ofautoantibodies against nuclear and endogenous antigens, among whichnucleosomes, DNA complexed to histones, seem to be the most prominent.

In vivo, nucleosomes are generated by apoptosis, a process that appearsdisturbed in lupus. In conditions of insufficient removal of apoptoticcells, nucleosomes act as autoantigens to drive a T cell-dependentimmune response, whose critical components are nucleosome-specificantibodies and nucleosome/IgG complexes. These bind to intrinsicconstituents of the glomerular basement membrane and promoteinflammation. Cytokines and chemoattractants generated in exuberantamount by renal resident and infiltrating cells amplify and perpetuateimmune complex-mediated injury.

Histologically, glomerular changes in NZB/W mice include endocapillaryhypercellularity associated with focal extracapillary proliferation;immune-type deposits are detected in the mesangium and on subendothelialaspect of the glomerular basement membrane (GBM). Tubular damage,interstitial inflammation and fibrosis are severe.

There is increasing evidence showing that positive (cyclins andcyclin-dependent kinases) and negative (cyclin-dependent kinaseinhibitors) cell cycle regulatory proteins have a critical role inregulating cellular responses to immune and nonimmune forms of injury,including cell proliferation and apoptosis. Proliferation of intrinsicglomerular cells such as mesangial cells is a characteristic response toforms of immune-mediated glomerular injury such as IgA nephropathy,lupus, and membranoproliferative glomerulonephritis. The expression ofpositive cell cycle proteins (cyclins D, E, A) as well as CDK2 proteinlevels and activity are increased in experimental mesangioproliferativeglomerulonephritis (Thy nephritis) characterized by mesangial cellproliferation. In this model, inhibiting CDK2 decreased mesangial cellproliferation and matrix protein accumulation and improved renalfunction.

In vivo studies by the applicant have shown that NZB/W F1 mice treatedwith CYC202 at the doses of 200 and 100 mg/kg, starting from 2 months ofage, survived significantly (P<0.05) longer than vehicle-treated mice.At the end of the study (month 8) while only four of thirteen NZB/W mice(31%) that had been treated with vehicle were alive, ten of thirteenmice (77%) and ten of fourteen mice (71%) treated with 200 and 100 mg/kgCYC202, respectively, survived. In the group of mice given CYC202 (100mg/kg) from 5 months of age (therapeutic treatment) the percentage ofsurvival was not different from that recorded in vehicle-treated mice.

In one preferred embodiment of the invention the amount of the inhibitorof CDK2 and/or CDK7 and/or CDK9 is sufficient to delay the onset ofproteinuria and renal function impairment.

Cumulative percentage of mice with heavy proteinuria (>4 mg/day) wasevaluated at different stages of the disease in all of the experimentalgroups. In the vehicle group the percentage of mice with proteinuriaprogressively increased over time. At the end of the study thepercentage of proteinuric mice was 85%. CYC202 given as a preventivetherapy from 2 months of age significantly delayed the onset ofproteinuria compared to vehicle, in a dose-dependent manner (%proteinuric mice, month 8: 200 mg/kg, 23%, P<0.01 vs vehicle; 100 mg/kg,43%, P<0.05 vs vehicle). When CYC202 was administered to lupus mice from5 months of age a tendency toward a reduced percentage of proteinuricmice in respect to vehicle was observed, which however did not reach thestatistical significance.

Renal function, assessed by serum blood urea nitrogen (BUN), wasmeasured at 5 and 8 months. At 5 months serum BUN levels were within thenormal range (<29 mg/dl) in all the experimental groups. In the vehiclegroup, renal function deteriorated with time and at 8 months 50% of thesurviving animals had BUN levels ≧30 mg/dl. CYC202 given as preventivetherapy from 2 months of age resulted in a better renal function oflupus mice, whereas it was not effective when administered at the laterage of 5 months.

In one preferred embodiment of the invention, the amount of theinhibitor of CDK2 and/or CDK7 and/or CDK9 is sufficient to down-regulatethe levels of anti-nuclear, specifically anti-DNA antibodies.

Elevated anti-DNA antibody levels are characteristic of NZB/W F1 mice.Mice given vehicle exhibited increasing levels of anti-DNA antibodiesover time. Either at 5 or 8 months of age, mice treated from 2 monthswith CYC202 at both doses showed anti-DNA antibody levels significantlylower than vehicle. In the group of mice receiving CYC202 from 5 monthsanti-DNA antibody concentration was numerically, albeit notsignificantly lower than vehicle at 8 months.

In one preferred embodiment, the inhibitor of CDK2 and/or CDK7 and/orCDK9 is in an amount sufficient to reduce glomerular andtubointerstitial changes.

At the end of the study NZB/W mice given vehicle revealed glomerularchanges with intracapillary hypercellularity associated with a focalextracapillary proliferation. Immune deposits were detected in themesangium and on the subendothelial aspect of the glomerular basementmembrane. Tubular damage and interstitial inflammation were alsoobserved. Treatment from 2 months of age with CYC202 markedly limitedglomerular hypercellularity, immune deposits, and tubulointerstitialdamage. These effects were more pronounced when CYC202 was given at thedose of 200 mg/kg. Only a mild effect on renal morphology was observedin mice administered CYC202 from 5 months of age.

In one preferred embodiment, the amount of the inhibitor of CDK2 and/orCDK7 and/or CDK9 is sufficient to inhibit T-cell proliferation inducedby PMA and ConA.

In one preferred embodiment of the invention, the inhibitor of CDK2and/or CDK7 and/or CDK9 is in an amount sufficient to down-regulate theexpression of Mcl-1.

In one preferred embodiment, the amount of the inhibitor of CDK2 and/orCDK7 and/or CDK9 is in an amount sufficient to reduce the interstitialaccumulation of mononuclear cells.

Kidneys were analysed for F4/80 positive monocytes/macrophages byimmunohistochemical technique. A marked accumulation of F4/80 positivecells was present in the renal interstitium of NZB/W mice given vehicle.Preventive treatment from 2 months of age with 200 mg/kg CYC202remarkably reduced the number of F4/80 positive monocytes/macrophages inrespect to vehicle. CYC202 at the dose of 100 mg/kg limited albeit notto a statistically significant degree the interstitial accumulation ofmononuclear cells. A numerical reduction of F4/80 positive cells wasobserved in the therapeutic study (treatment from 5 months of age).

By way of summary, the results of the present study clearly indicatethat CYC202 (200 and 100 mg/kg) given as a preventive therapy from 2months of age, retarded renal manifestation of lupus in NZB/W mice andremarkably prolonged life as compared to animals given vehicle.Specifically, CYC202 delayed the onset of proteinuria and renal functionimpairment, and limited glomerular and tubulointerstitial changesincluding the interstitial accumulation of mononuclear cells, glomerularhypercellularity and immune deposits. The effects are more pronounced atthe dose of 200 mg/kg. A remarkable finding of the present study was thereduction of the levels of anti-DNA antibodies by CYC202, which couldpossibly be attributed to CYC202 effects on T cells, which in turn mayaffect B cells. By in vitro experiments, a concentration-dependentinhibition of T cell proliferation induced by PMA and ConA, as well asin mixed lymphocyte reaction, has been observed following treatment withCYC202. Furthermore, there is evidence that in SLE, activated autoimmuneT helper cells specific for histones or nucleosomes may provide help forB cells to differentiate into anti-DNA producing plasma cells (forreview see Rekvig, Arthritis & Rheumatism 48: 300-312, 2003).

CYC202 is also known to have an effect on transcription, includingdownregulation of the anti-apoptotic protein mcl-1 and is thus capableof altering the balance of survival signals in cells. Anergic T cellsmay be specifically sensitive to this mechanism of action.

The administration of CYC202 (100 mg/kg) initiated at 5 months of ageresulted in a mild reduction of the percentage of proteinuric mice andof renal damage in respect to vehicle-mice. Survival was notameliorated. The modest effect was probably due to the low dose.

Combinations

In one particularly preferred embodiment, the medicament is for use incombination therapy with methylprednisolone.

As used herein, the term “combination therapy” refers to therapy inwhich the methylprednisolone and the inhibitor of CDK2 and/or CDK7and/or CDK9, are administered, if not simultaneously, then sequentiallywithin a timeframe that they both are available to act therapeuticallywithin the same time-frame.

Another aspect of the invention relates to a combination comprising aninhibitor of CDK2 and/or CDK7 and/or CDK9, or a pharmaceuticallyacceptable salt thereof, and methylprednisolone.

Preferably, the combination has a synergistic effect, i.e. thecombination is synergistic. Methylprednisolone is a synthetic (man-made)corticosteroid. The chemical name is11,17,21-trihydroxy-6-methyl-(6a,11b)-pregna-1,4-diene-3,20-dione.Corticosteroids are naturally-occurring chemicals produced by theadrenal glands located adjacent to the kidneys. Corticosteroids blockinflammation and are used in a wide variety of inflammatory diseases.There are numerous preparations of corticosteroids including oraltablets, capsules, liquids, topical creams and gels, inhalers, eyedrops, and injectable and intravenous solutions. Methylprednisolone istypically prescribed as an oral tablet or liquid.

Methylprednisolone is used to achieve prompt suppression ofinflammation. Examples of inflammatory conditions for whichmethylprednisolone is used include rheumatoid arthritis, systemic lupuserythmatosus, acute gouty arthritis, psoriatic arthritis, ulcerativecolitis, and Crohn's disease. Severe allergic conditions that failconventional treatment also may respond to methylprednisolone. Examplesinclude bronchial asthma, allergic rhinitis, drug-induced dermatitis,and contact and atopic dermatitis. Chronic skin conditions treated withmethylprednisolone include dermatitis herpetiformis, pemphigus, severepsoriasis and severe seborrheic dermatitis. Chronic allergic andinflammatory conditions of the uvea, iris, conjunctiva and optic nervesof the eyes also are treated with methylprednisolone.

Another aspect relates to a pharmaceutical composition comprising acombination according to the invention and a pharmaceutically acceptablecarrier, diluent or excipient.

Another aspect of the invention relates to a pharmaceutical productcomprising an inhibitor of CDK2 and/or CDK7 and/or CDK9, or apharmaceutically acceptable salt thereof, and methylprednisolone as acombined preparation for simultaneous, sequential or separate use intherapy.

The inhibitor of CDK2 and/or CDK7 and/or CDK9 and methylprednisolone maybe administered simultaneously, in combination, sequentially orseparately (as part of a dosing regime).

As used herein, “simultaneously” is used to mean that the two agents areadministered concurrently, whereas the term “in combination” is used tomean they are administered, if not simultaneously, then “sequentially”within a timeframe that they both are available to act therapeuticallywithin the same time-frame. Thus, administration “sequentially” maypermit one agent to be administered within 5 minutes, 10 minutes or amatter of hours after the other provided the circulatory half-life ofthe first administered agent is such that they are both concurrentlypresent in therapeutically effective amounts. The time delay betweenadministration of the components will vary depending on the exact natureof the components, the interaction therebetween, and their respectivehalf-lives.

In contrast to “in combination” or “sequentially”, “separately” is usedherein to mean that the gap between administering one agent and theother is significant i.e. the first administered agent may no longer bepresent in the bloodstream in a therapeutically effective amount whenthe second agent is administered.

Yet another aspect of the invention relates to a pharmaceuticalcomposition comprising:

-   (i) an inhibitor of CDK2 and/or CDK7 and/or CDK9, or a    pharmaceutically acceptable salt thereof; and-   (ii) methylprednisolone;    admixed with a pharmaceutically acceptable diluent, excipient or    carrier.

A further aspect relates to a combination according to the invention inthe preparation of a medicament for treating a disease associated withantinuclear antibodies.

A further aspect of the invention relates to the use of an inhibitor ofCDK2 and/or CDK7 and/or CDK9, or a pharmaceutically acceptable saltthereof, in the preparation of a medicament for treating a diseaseassociated with antinuclear antibodies, wherein the medicament is foruse in combination with methylprednisolone.

Yet another aspect of the invention relates to the use ofmethylprednisolone in the preparation of a medicament for treating adisease associated with antinuclear antibodies, wherein the medicamentis for use in combination with an inhibitor of CDK2 and/or CDK7 and/orCDK9, or a pharmaceutically acceptable salt thereof.

A further aspect of the invention relates to the use of an inhibitor ofCDK2 and/or CDK7 and/or CDK9, or a pharmaceutically acceptable saltthereof, and methylprednisolone, in the preparation of a medicament fortreating a disease associated with antinuclear antibodies.

Another aspect of the invention relates to the use of an inhibitor ofCDK2 and/or CDK7 and/or CDK9, or a pharmaceutically acceptable saltthereof, in the preparation of a medicament for the treatment of adisease associated with antinuclear antibodies, wherein said treatmentcomprises administering to a subject simultaneously, sequentially orseparately an inhibitor of CDK2 and/or CDK7 and/or CDK9, or apharmaceutically acceptable salt thereof, and methylprednisolone.

A further aspect of the invention relates to a method of treating adisease associated with antinuclear antibodies in a subject, said methodcomprising administering to the subject a therapeutically acceptableamount of:

-   (i) an inhibitor of CDK2 and/or CDK7 and/or CDK9, or a    pharmaceutically acceptable salt thereof; and-   (ii) methylprednisolone.

For all of the above embodiments, preferably, the inhibitor of CDK2and/or CDK7 and/or CDK9 and methylprednisolone are administeredsimultaneously or sequentially.

In one preferred embodiment, the inhibitor of CDK2 and/or CDK7 and/orCDK9 and methylprednisolone are administered simultaneously.

In one particularly preferred embodiment, the inhibitor of CDK2 and/orCDK7 and/or CDK9, is administered to the subject prior to sequentiallyor separately administering methylprednisolone to said subject.

Another aspect of the invention relates to a method of treating aproliferative disorder comprising the sequential administration of atherapeutically effective amount of an inhibitor of CDK2 and/or CDK7and/or CDK9, followed by a therapeutically effective amount ofmethylprednisolone.

Another aspect of the invention relates to the use of an inhibitor ofCDK2 and/or CDK7 and/or CDK9 in the manufacture of a medicament for usein the treatment of proliferative disorders comprising the sequentialadministration of a therapeutically effective amount of an inhibitor ofCDK2 and/or CDK7 and/or CDK9, followed by a therapeutically effectiveamount of methylprednisolone.

In an alternative preferred embodiment, methylprednisolone isadministered to the subject prior to sequentially or separatelyadministering the inhibitor of CDK2 and/or CDK7 and/or CDK9 to saidsubject.

In one particularly preferred embodiment, the inhibitor of CDK2 and/orCDK7 and/or CDK9 and methylprednisolone are administered sequentially.

In one preferred embodiment of the invention, the inhibitor of CDK2and/or CDK7 and/or CDK9 and methylprednisolone are each administered ina therapeutically effective amount with respect to the individualcomponents.

In another preferred embodiment of the invention, the inhibitor of CDK2and/or CDK7 and/or CDK9 and methylprednisolone are each administered ina subtherapeutic amount with respect to the individual components.

Preferably, the inhibitor of CDK2, CDK7 or CDK9 is as described abovefor the first aspect of the invention.

Pharmaceutical Compositions

As mentioned above, various aspects of the invention relate topharmaceutical compositions.

Even though the compounds of the present invention (including theirpharmaceutically acceptable salts, esters and pharmaceuticallyacceptable solvates) can be administered alone, they will generally beadministered in admixture with a pharmaceutical carrier, excipient ordiluent, particularly for human therapy. The pharmaceutical compositionsmay be for human or animal usage in human and veterinary medicine.

Examples of such suitable excipients for the various different forms ofpharmaceutical compositions described herein may be found in the“Handbook of Pharmaceutical Excipients”, 2^(nd) Edition, (1994), Editedby A Wade and P J Weller.

Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).

Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol and the like. Examplesof suitable diluents include ethanol, glycerol and water.

The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as, or in addition to, the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Examples of suitable binders include starch, gelatin, natural sugarssuch as glucose, anhydrous lactose, free-flow lactose, beta-lactose,corn sweeteners, natural and synthetic gums, such as acacia, tragacanthor sodium alginate, carboxymethyl cellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like.

Preservatives, stabilizers, dyes and even flavoring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

Salts/Esters

The compounds used in the invention can be present as salts or esters,in particular pharmaceutically acceptable salts or esters.

Pharmaceutically acceptable salts of the compounds of the inventioninclude suitable acid addition or base salts thereof. A review ofsuitable pharmaceutical salts may be found in Berge et al, J Pharm Sci,66, 1-19 (1977). Salts are formed, for example with strong inorganicacids such as mineral acids, e.g. sulphuric acid, phosphoric acid orhydrohalic acids; with strong organic carboxylic acids, such asalkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted orsubstituted (e.g., by halogen), such as acetic acid; with saturated orunsaturated dicarboxylic acids, for example oxalic, malonic, succinic,maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylicacids, for example ascorbic, glycolic, lactic, malic, tartaric or citricacid; with aminoacids, for example aspartic or glutamic acid; withbenzoic acid; or with organic sulfonic acids, such as (C₁-C₄)-alkyl- oraryl-sulfonic acids which are unsubstituted or substituted (for example,by a halogen) such as methane- or p-toluene sulfonic acid.

Esters are formed either using organic acids or alcohols/hydroxides,depending on the functional group being esterified. Organic acidsinclude carboxylic acids, such as alkanecarboxylic acids of 1 to 12carbon atoms which are unsubstituted or substituted (e.g., by halogen),such as acetic acid; with saturated or unsaturated dicarboxylic acid,for example oxalic, malonic, succinic, maleic, fumaric, phthalic ortetraphthalic; with hydroxycarboxylic acids, for example ascorbic,glycolic, lactic, malic, tartaric or citric acid; with aminoacids, forexample aspartic or glutamic acid; with benzoic acid; or with organicsulfonic acids, such as (C₁-C₄)-allyl- or aryl-sulfonic acids which areunsubstituted or substituted (for example, by a halogen) such asmethane- or p-toluene sulfonic acid. Suitable hydroxides includeinorganic hydroxides, such as sodium hydroxide, potassium hydroxide,calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcoholsof 1-12 carbon atoms which may be unsubstituted or substituted, e.g. bya halogen).

Enantiomers/Tautomers

In all aspects of the present invention previously discussed, theinvention includes the use of, where appropriate, all enantiomers andtautomers of the compounds involved. The man skilled in the art willrecognise compounds that possess an optical properties (one or morechiral carbon atoms) or tautomeric characteristics. The correspondingenantiomers and/or tautomers may be isolated/prepared by methods knownin the art.

Stereo and Geometric Isomers

The compounds used in the invention may exist as stereoisomers and/orgeometric isomers—e.g. they may possess one or more asymmetric and/orgeometric centres and so may exist in two or more stereoisomeric and/orgeometric forms. The present invention contemplates the use of all theindividual stereoisomers and geometric isomers of those agents, andmixtures thereof. The terms used in the claims encompass these forms,provided said forms retain the appropriate functional activity (thoughnot necessarily to the same degree).

The present invention also includes the use of all suitable isotopicvariations of the agent or pharmaceutically acceptable salt thereof. Anisotopic variation of an agent of the present invention or apharmaceutically acceptable salt thereof is defined as one in which atleast one atom is replaced by an atom having the same atomic number butan atomic mass different from the atomic mass usually found in nature.Examples of isotopes that can be incorporated into the agent andpharmaceutically acceptable salts thereof include isotopes of hydrogen,carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorinesuch as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶Cl,respectively. Certain isotopic variations of the agent andpharmaceutically acceptable salts thereof, for example, those in which aradioactive isotope such as 3H or ¹⁴C is incorporated, are useful indrug and/or substrate tissue distribution studies. Tritiated, i.e., ³H,and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for theirease of preparation and detectability. Further, substitution withisotopes such as deuterium, i.e., ²H, may afford certain therapeuticadvantages resulting from greater metabolic stability, for example,increased in vivo half-life or reduced dosage requirements and hence maybe preferred in some circumstances. Isotopic variations of the agent ofthe present invention and pharmaceutically acceptable salts thereof ofthis invention can generally be prepared by conventional proceduresusing appropriate isotopic variations of suitable reagents.

Solvates

The present invention also includes the use of solvate forms of thecompounds of the present invention. The terms used in the claimsencompass these forms.

Polymorphs

The invention furthermore relates to the use of compounds of the presentinvention in their various crystalline forms, polymorphic forms and(an)hydrous forms. It is well established within the pharmaceuticalindustry that chemical compounds may be isolated in any of such forms byslightly varying the method of purification and or isolation form thesolvents used in the synthetic preparation of such compounds.

Prodrugs

The invention further includes the ues of compounds of the presentinvention in prodrug form. Such prodrugs are generally compounds whereinone or more appropriate groups have been modified such that themodification may be reversed upon administration to a human or mammaliansubject. Such reversion is usually performed by an enzyme naturallypresent in such subject, though it is possible for a second agent to beadministered together with such a prodrug in order to perform thereversion in vivo. Examples of such modifications include ester (forexample, any of those described above), wherein the reversion may becarried out be an esterase etc. Other such systems will be well known tothose skilled in the art.

Administration

The pharmaceutical compositions of the present invention may be adaptedfor oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal,intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal,intravenous, nasal, buccal or sublingual routes of administration.

For oral administration, particular use is made of compressed Tablets,pills, Tablets, gellules, drops, and capsules. Preferably, thesecompositions contain from 1 mg to 5000 mg and more preferably from 10mg-3000 mg, of active ingredient per dose.

Other forms of administration comprise solutions or emulsions which maybe injected intravenously, intraarterially, intrathecally,subcutaneously, intradermally, intraperitoneally or intramuscularly, andwhich are prepared from sterile or sterilizable solutions. Thepharmaceutical compositions of the present invention may also be in formof suppositories, pessaries, suspensions, emulsions, lotions, ointments,creams, gels, sprays, solutions or dusting powders.

An alternative means of transdermal administration is by use of a skinpatch. For example, the active ingredient can be incorporated into acream consisting of an aqueous emulsion of polyethylene glycols orliquid paraffin. The active ingredient can also be incorporated, at aconcentration of between 1 and 10% by weight, into an ointmentconsisting of a white wax or white soft paraffin base together with suchstabilisers and preservatives as may be required.

Injectable forms may contain between 10-3000 mg, preferably between10-1000 mg, of active ingredient per dose.

Compositions may be formulated in unit dosage form, i.e., in the form ofdiscrete portions containing a unit dose, or a multiple or sub-unit of aunit dose.

Dosage

A person of ordinary skill in the art can easily determine anappropriate dose of one of the instant compositions to administer to asubject without undue experimentation. Typically, a physician willdetermine the actual dosage which will be most suitable for anindividual patient and it will depend on a variety of factors includingthe activity of the specific compound employed, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and theindividual undergoing therapy. The dosages disclosed herein areexemplary of the average case. There can of course be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention.

Depending upon the need, the agent may be administered at a dose of from0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, morepreferably from 0.1 to 1 mg/kg body weight.

In an exemplary embodiment, one or more doses of 10 to 3500 mg/day willbe administered to the patient.

The present invention is further illustrated by way of example, and withreference to the following Figures wherein:

FIG. 1 shows the percentage survival against the duration of treatment(months) for NZB/W F1 mice treated with CYC202 at dosages of 100 and 200mg/kg from 2 months or 5 months.

FIG. 2 shows the percentage proteinuric mice against the duration oftreatment (months) for NZB/W F1 mice treated with CYC202 at dosages of100 mg/kg or 200 mg/kg.

FIG. 3 shows serum anti-DNA antibodies (U/ml) for NZB/W F1 mice treatedwith CYC202 at 100 mg/kg or 200 mg/kg (versus control vehicle).

FIG. 4 shows BrdU incorporation (top) and viability (bottom) ofunstimulated and stimulated PBMCs and control cells, untreated ortreated with DMSO vehicle.

FIG. 5 shows the difference in BrdU incorporation between PBMC cellsstimulated with PHA or PMA/I for 48 hour, when treated with DMSO (topleft) or compounds at 4×IC50, 3×IC₅₀, 2×IC₅₀ and IC₅₀ concentrations, 2hours before stimulation (−2 h), at the time of stimulation (0 h) and 2hours after stimulation (+2 h).

FIG. 6 shows the difference in viability between PBMC cells stimulatedwith PHA or PMA/I for 48 hour, when treated with DMSO (top left) orcompounds at 4×IC₅₀, 3×IC₅₀, 2×IC₅₀ and IC₅₀ concentrations, 2 hoursbefore stimulation (−2 h), at the time of stimulation (0 h) and 2 hoursafter stimulation (+2 h).

FIG. 7 shows the titration of Concanavalin A shows maximum stimulationwith 10 μg/mL (top left), and reduced viability at higher concentrations(top right). Increase in BrdU incorporation is comparable with PHA andPMA/I stimulation (bottom left).

FIG. 8 shows BrdU incorporation (top) and viability (bottom) in PBMCsstimulated with PHA and ConA and unstimulated cells, at 48 and 72 hourstreatment with DMSO and untreated.

FIG. 9 shows BrdU incorporation in PHA (left) and ConA (right)stimulated PBMCs treated with compounds at 4×IC₅₀, 3×IC₅₀, 2×IC₅₀, IC₅₀,0.5×IC₅₀ and 0.25×IC₅₀ and DMSO control, for 48 and 72 hours.

FIG. 10 shows the viability of PHA (left) and ConA (right) stimulatedPBMCs treated with compounds at 4×IC₅₀, 3×IC₅₀, 2×IC₅₀, IC₅₀, 0.5×IC₅₀and 0.25×IC₅₀ and DMSO control, for 48 and 72 hours.

EXAMPLES Inhibitors of CDK2 and/or CDK7 and/or CDK9

Various inhibitor compounds were prepared in accordance with the methodsdescribed in EP 0874847B (CNRS); and WO 03/002565, WO 04/016613, WO04/016612, WO01/72745, WO 02/079193, WO 03/029248, WO04/043953 (all inthe name of Cyclacel Limited).

Kinase Assays

Kinase activity was investigated by measuring the incorporation ofradioactive phosphate from ATP into appropriate polypeptide substrates.Recombinant protein kinases and kinase complexes were produced orobtained commercially. Assays were performed using 96-well plates andappropriate assay buffers (typically 25 mM β-glycerophosphate, 20 mMMOPS, 5 mM EGTA, 1 mM DTT, 1 mM Na₃VO₃, pH 7.4), into which were added2-4 μg of active enzyme with appropriate substrates. The reactions wereinitiated by addition of Mg/ATP mix (15 mM MgCl₂+100 μM ATP with 30-50kBq per well of [γ-³²P]-ATP) and mixtures incubated as required at 30°C. Reactions were stopped on ice, followed by filtration through p81filterplates or GF/C filterplates (Whatman Polyfiltronics, Kent, UK).After washing 3 times with 75 mM aq orthophosphoric acid, plates weredried, scintillant added and incorporated radioactivity measured in ascintillation counter (TopCount, Packard Instruments, Pangbourne, Berks,UK). Compounds for kinase assay were made up as 10 mM stocks in DMSO anddiluted into 10% DMSO in assay buffer. Data was analysed usingcurve-fitting software (GraphPad Prism version 3.00 for Windows,GraphPad Software, San Diego Calif. USA) to determine IC₅₀ values(concentration of test compound which inhibits kinase activity by 50%).

CDK 7 and 9 Assay

CTD peptide substrate (biotinyl-Ahx-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)₄-NH₂;1-2 mg/mL) and recombinant human CDK7/cyclin H, CDK9/cyclin T1, orCDK9/cyclin K (0.5-2 μg) were incubated for 45 min at 30° C. in thepresence of varying amounts of test compound in 20 mM MOPS pH 7.2, 25 mMβ-glycerophosphate, 5 mM EGTA, 1 mM DTT, 1 mM sodium vanadate, 15 mMMgCl₂, and 100 μM ATP (containing a trace amount of ³²PγATP) in a totalvolume of 25 μL in a 96-well microtiter plate. The reaction was stoppedby placing the plate on ice for 2 min. Avidin (50 μg) was added to eachwell, and the plate was incubated at room temp for 30 min. The sampleswere transferred to a 96-well P81 filter plate, and washed (4×200 μL perwell) with 75 mM phosphoric acid. Microscint 40 scintillation liquid (50μL) was added to each well, and the amount of ³²P incorporation for eachsample was measured using a Packard Topcount microplate scintillationcounter.

CYC202

Studies were undertaken to evaluate whether administration of the CDK2inhibitor CYC202 was effective in retarding the development of renaldisease of NZB/WF1 lupus prone mice.

NZBxNZW F 1 female mice (Harlan Italy s.r.l., Milano, Italy) of 2 monthsof age at the start of the experiment, were used. Animal care andtreatment were conducted in accordance with the institutional guidelinesthat are in compliance with national (Decreto Legislativo n.116,Gazzetta Ufficiale suppl 40, 18 febbraio 1992, Circolare n.8, GazzettaUfficiale 14 luglio 1994) and international laws and policies (EECCouncil Directive 86/609, OJL358-1, December 1987; Guide for the Careand Use of Laboratory Animals, U.S. National Research Council, 1996).Animals were housed in a constant temperature room with a 12-hourdark/12-hour light cycle and were fed a standard diet.

Example 1

NZBxNZW F1 mice were randomly allocated to the following groups:

Group 1 (n=18): mice that were given daily by gavage the vehicle (HCl 50mM; Group 2 (n=17): mice that were given daily by gavage CYC202 (200mg/kg); Group 3 (n=19): mice that were given daily by gavage CYC202 (100mg/kg); Treatments started at 2 months of age (preventive study) andlasted until 8 months; Four-five animals of each group were sacrified at5 months for evaluation of serum BUN and levels of circulating anti-DNAantibodies.

An additional group, Group 4 (n=14 mice) was given daily by gavageCYC202 (100 mg/kg) starting at 5 months of age, a time when immunecomplex deposition is actively taking place, until 8 months (therapeuticstudy). Five normal CD-1 mice (Charles River Italia, Calco, Italy) wereused as control.

The following parameters were evaluated:

Urinary protein excretion: determined every month until 5 months of ageand then every two weeks.

At sacrifice:

Anti-DNA antibodies in the serum; Serum BUN; Serum transaminase (AST,ALT); Renal histology; Accumulation of monocytes/macrophages into therenal interstitium. Example 2

Lupus mice were randomly allocated to the following groups:

Group 1 (n=10): mice that were given daily by gavage the vehicle (HCl 50mM); Group 2 (n=15): mice that were given daily by gavage CYC202 at thedose of 200 mg/kg; Group 3 (n=12): mice that were given daily byintraperitoneal injection methylprednisolone (MPS, Urbason, Hoechsts.p.a, Milano, Italy) at the dose of 1.5 mg/kg; Group 4 (n=16): micethat were given daily CYC202 (200 mg/kg) in combination with MPS (1.5mg/kg).

Treatments started at 5 months of age, a time when immunecomplexdeposition is actively taking place and lasted until 12 months when thelast animal receiving the vehicle treatment died. Five normal CD-1 mice(Charles River Italia, Calco, Italy) were used as control.

The following parameters were evaluated:

Survival;

Urinary protein excretion: determined every month until 5 months of ageand then every two weeks;

Serum BUN: measured at month 5 (before treatment) and every month untilthe end of the study,

Renal histology: evaluated in biopsies from mice that were terminallyill and from mice survived until 12 months;

Accumulation of F4/80 positive monocytes/macrophages into the renalinterstitium (evaluated in the same biopsies as above). Examples 1 and 2Materials and Methods Proteinuria and Renal Function

Urinary protein concentration was determined by the Coomassie blue Gdye-binding assay with bovine serum albumin as standard. Renal functionwas assessed as BUN in heparinized blood by the Reflotron test (RocheDiagnostics Corporation, Indianapolis, USA). BUN levels exceeding 30mg/dl were considered abnormal (normal range in this laboratory formice: 14-29 mg/dl).

Anti-DNA Antibodies

The levels of anti-dsDNA autoantibodies were evaluated in the serum byan enzyme-immunoassay (Diastat anti-ds DNA kit, Bouty Laboratory,Milano, Italy) as described before (Kidney Int, 53:726-734, 1998).

Serum Transaminase

Serum levels of AST and ALT were measured using an autoanalyzer (CX5,Beckman Instruments Inc., Fullerton, Calif.).

Renal Morphology

Light microscopy: Fragments of renal cortex were fixed in Dubosq-Brazil,dehydrated in alcohol and embedded in paraffin. Sections (3 μm) werestained with hematoxylin and eosin, Masson's trichrome, and periodicacid-Schiff's reagent (PAS-stain). Glomerular intracapillaryhypercellularity was evaluated in a semiquantitative fashion by ascoring system from 0 to 3+(0=no hypercellularity; 1+=mild; 2+=moderate;3+=severe). A single score was given for other changes based on thepercentages of total glomeruli involved with a lesion. Extracapillaryproliferation was graded from 0 to 3+(0=no hypercellularity; 1+=lessthan 25% of glomeruli involved; 2+=25% to 50% of glomeruli involved;3+=more than 50% of glomeruli involved). Glomerular deposits were gradedfrom 0 to 3+(0=no deposits; 1+=less than 25% of glomeruli involved;2+=25% to 50% of glomeruli involved; 3+=more than 50% of glomeruliinvolved). Tubular (atrophy, casts and dilatation) and interstitialchanges (fibrosis and inflammation) were graded from 0 to 3+(0=nochanges; 1+=changes affecting less than 25% of the sample; 2+=changesaffecting 25 to 50% of the sample; 3+=changes affecting more than 50% ofthe sample). At least 100 glomeruli were examined for each biopsy. Atleast 10 fields per sample were examined at low magnification (10×) forhistologic scoring of the interstitium. All renal biopsies have beenanalyzed by the same pathologist, in a single-blind fashion.

Immunohistochemical Analysis

Rat monoclonal antibody against a cytoplasmic antigen present in mousemonocytes and macrophages (F4/80, 4 μg/ml, Caltag Laboratories,Burlingame, Calif.) was used for the detection of infiltrating cells byimmunoperoxidase technique. Sections were incubated for 30 minutes with0.3% H2O2 in methanol to quench endogenous peroxidase. Then the tissuewas permeabilized in 0.1% Triton X-100 in PBS 0.01 mol/L, PH 7.2, for 30minutes and then incubated with normal goat serum (Vector Laboratories)for 30 minutes. Primary antibody was incubated overnight at 4° C.,followed by the secondary antibody (biotinylated goat anti-rat IgG,Vector Laboratories) and avidin-biotin peroxidase complex (ABC)solution, and finally development with DAB. The sections werecounterstained with Harris hematoxylin. Negative controls were obtainedby omitting the primary antibody. F40/80 labelled cells were counted inat least 10 randomly selected high power microscopic fields (×400) pereach animal.

Statistical Analysis

Data are expressed as mean ±Standard Error (SE). Survival curves wereanalyzed by log-rank test. Proteinuria data were analyzed by Fisher'sexact test. All the other parameters were analyzed by Kruskall Wallistest Statistical significance was defined as P<0.05.

Example 1 Results Body Weight, Food and Water Intake

As shown in Table 1 lupus mice gained weight during the study. Nodifference in body weight was observed among the experimental groups.Food (Table 2) and water (Table 3) intake evaluated every two weeks from2 to 5 months were comparable among vehicle and CYC202 treated mice.

Lupus Mice Survival

NZB/W F1 mice treated with CYC202 at the doses of 200 and 100 mg/kg,starting from 2 months of age, survived significantly (P<0.05) longerthan vehicle-mice (see Table 4 and FIG. 1). Actually, at the end of thestudy (month 8) while only four of thirteen NZB/W mice (31%) that hadbeen treated with vehicle were alive, ten of thirteen mice (77%) and tenof fourteen mice (71%) treated with 200 and 100 mg/kg CYC202,respectively, survived. In the group of mice given CYC202 (100 mg/kg)from 5 months of age (therapeutic treatment) the percentage of survivalwas not different from that recorded in vehicle-mice.

Proteinuria and Renal Function

Cumulative percentage of mice with heavy proteinuria (>4 mg/day) wasevaluated at different stages of the disease in all of the experimentalgroups. As shown in Table 5, in the vehicle group the percentage of micewith proteinuria progressively increased over time (FIG. 2). At the endof the study the percentage of proteinuric mice was 85%. CYC202 given asa preventive therapy significantly delayed the onset of proteinuriacompared to vehicle, in a dose-dependent manner (% proteinuric mice,month 8: 200 mg/kg, 23%, P<0.01 vs vehicle; 100 mg/kg, 43%, P<0.05 vsvehicle). When CYC202 was administered to lupus mice from 5 months ofage a tendency toward a reduced percentage of proteinuric mice inrespect to vehicle was observed, which however did not reach thestatistical significance.

Renal function, assessed by serum BUN, was measured at 5 and 8 months.At 5 months serum BUN levels were within the normal range (<29 mg/dl) inall the experimental groups. In the vehicle group, renal functiondeteriorated with time and at 8 months 50% of the surviving animals hadBUN levels ≧30 mg/dl (Table 6). CYC202 given as preventive therapyresulted in a better renal function of lupus mice, whereas it was noteffective when administered late.

Anti-DNA Antibodies

Elevated anti-DNA antibody levels are characteristic of NZB/W F1 mice.As shown in Table 7, mice given vehicle exhibited increasing levels ofanti-DNA antibodies over time. Either at 5 or 8 months of age, micetreated from 2 months with CYC202 at both doses showed anti-DNA antibodylevels significantly lower than vehicle. In the group of mice receivingCYC202 from 5 months anti-DNA antibody concentration was numerically,albeit not significantly lower than vehicle at 8 months (FIG. 3).

Serum Transaminase Levels

Serum ALT and AST levels were measured in NZB/W F1 mice given 200 mg/kgCYC202 from 2 months or 100 mg/kg CYC202 from 5 months. Serumtransaminase levels were not modified by treatments and values weresimilar to those of control mice (Table 8).

Renal Morphology

As shown in Table 9, at the end of the study NZB/W mice given vehiclerevealed glomerular changes with intracapillary hypercellularityassociated with a focal extracapillary proliferation. Immune type ofdeposits were detected in the mesangium and on subendothelial aspect ofthe glomerular basement membrane. Tubular damage and interstitialinflammation were also observed. Treatment from 2 months with CYC202markedly limited glomerular hypercellularity, immune deposits, andtubulointerstitial damage. These effects were more pronounced whenCYC202 was given at the dose of 200 mg/kg. Only a mild effect on renalmorphology was observed in mice administered CYC202 from 5 months.

Interstitial Accumulation of Monocytes/Macrophages

Kidneys were analysed for F4/80 positive monocytes/macrophages byimmunohistochemical technique. A marked accumulation of F4/80 positivecells was present in the renal interstitium of NZB/W mice given vehicle(Table 10). Preventive treatment with 200 mg/kg CYC202 remarkablyreduced the number of F4/80 positive monocytes/macrophages in respect tovehicle. CYC202 at the dose of 100 mg/kg limited albeit not to astatistically significant degree the interstitial accumulation ofmononuclear cells. A numerical reduction of F4/80 positive cells wasobserved in the therapeutic study.

The results of the present project clearly indicate that CYC202 (200 and100 mg/kg) given as a preventive therapy from 2 months of age, retardedrenal manifestation of lupus in NZB/W mice and remarkably prolonged lifeas compared to animals given vehicle. Specifically, CYC202 delayed theonset of proteinuria and renal function impairment, and limitedglomerular and tubulointerstitial changes including the interstitialaccumulation of mononuclear cells, the effects being more pronounced atthe dose of 200 mg/kg. A remarkable finding of the present study was thereduction of the levels of anti-DNA antibodies by CYC202, which couldpossibly be attributed to CYC202 effects on T cells which in turn mayaffect B cells. By in vitro experiments a concentration-dependentinhibition of T cell proliferation induced by PMA and ConA as well as inmixed lymphocyte reaction has been observed. On the other hand there isevidence that in SLE, activated autoimmune T cells specific for histonesor nucleosomes may provide help for B cells to differentiate intoanti-DNA producing plasma cells (for review see Rekvig, Arthritis &Rheumatism 48: 300-312, 2003).

The administration of CYC202 (100 mg/kg) initiated at 5 months of ageresulted in a mild reduction of the percentage of proteinuric mice andof renal damage in respect to vehicle-mice. Survival was notameliorated.

Example 2 Results Body Weight

NZB/W F1 mice gained weight during the study. No difference in bodyweight was observed among the experimental groups.

Survival

NZB/W F1 mice treated with the combination of CYC202 andmethylprednisolone (MPS), starting from 5 months of age, survivedsignificantly (P<0.0001) longer than vehicle-mice (Table 11). Notably,at 12 months, when all mice given vehicle died, ten of sixteen animals(62%) treated with the combined therapy were alive. Survival curves ofmice receiving the single therapies were not different from that ofvehicle group.

Proteinuria

Table 12 shows the cumulative percentage of mice with proteinuria >4mg/day evaluated at different stages of the disease. The association ofCYC202 and MPS significantly delayed the onset of proteinuria comparedto vehicle. In the interval from 7 to 10 months the proportion ofproteinuric mice in the combined therapy group was significantly lowerthan in the vehicle group (6.2 to 43.8% versus 40 to 90%). CYC202 or MPSadministered as single therapies only partially affected the onset ofproteinuria as compared to vehicle, with a significant reduction in thepercentage of proteinuric mice being observed at 7.5 months for theCYC202 group.

Renal Function

Renal function, assessed by serum BUN, was measured monthly from 5(before treatment) to 12 months. Table 13 shows the cumulativepercentage of mice with BUN levels ≧30 mg/dl. At 5 months serum BUNlevels were within the normal range (i.e. 14 to 29 mg/dl) in all theexperimental groups. In the vehicle group, renal function deterioratedwith time. Thus, at 8 and 12 months 80% and 100% of animals,respectively, had BUN levels ≧30 mg/dl. By contrast, only 27% and 53% ofmice on the combined therapy had impaired renal function at these timepoints. CYC202 or MPS given alone displayed a less renoprotective effectthan when used in combination.

Renal Morphology

Morphological analysis was performed on renal specimens taken fromeither terminally ill mice at different times or mice sacrified at 12months. Data are reported in Table 14. NZB/W mice given vehicle revealedglomerular changes with intracapillary hypercellularity associated witha focal extracapillary proliferation. Immune-type deposits were detectedin the mesangium and on the subendothelial aspect of the glomerularbasement membrane. Tubular damage and interstitial inflammation werealso observed. Treatment with CYC202 plus MPS markedly limitedglomerular hypercellularity, immune deposits, and tubulointerstitialdamage. A mild effect on renal morphology was observed in miceadministered CYC202 or MPS alone.

Interstitial Accumulation of Monocytes/Macrophages

Kidneys were analysed for F4/80 positive monocytes/macrophages byimmunohistochemical technique. A marked accumulation of F4/80 positivecells was present in the renal interstitium of NZB/W mice given vehicle(Table 15). The combined administration of CYC202 and MPS resulted in65% reduction of the number of monocytes/macrophages in respect tovehicle (P<0.01). CYC202 given alone reduced interstitial infiltrates by33%. In the group of mice treated with MPS alone interstitialaccumulation of mononuclear cells was similar to that found in thevehicle group.

By way of summary, the results showed that CYC202 combined with low dosemethylprednisolone significantly prolonged the lupus mice lifespan.Notably, the treatment was initiated at a phase of established disease,i.e. 5 months of age, when immune complex deposition is actively takingplace. The combined therapy delayed the onset of proteinuria, limitedimpairment of renal function and development of glomerularhypercellularity, immune deposits and tubulointerstitial changes. Ananti-inflammatory effect was also displayed, as indicated by a reducedaccumulation of mononuclear cells into the renal interstitium. Theadministration of CYC202 or methylprednisolone as single therapiesresulted in a mild renoprotective effect.

In conclusion, the results of the therapeutic effect of combined CYC202and low dose MPS in ameliorating the renal manifestation of lupus andprolonging survival in lupus mice with established disease couldrepresent the basis for a new treatment for lupus nephritis.

Examples 3 and 4 Materials and Methods

Selected CDK 2, 7 and 9 inhibitors (transcriptional inhibitors),compounds [1]-[12] were tested in a T cell proliferation assay, chosenas a surrogate assay for effect on anti-nuclear antibodies.

As mentioned above, anti-nuclear antibody production can only be studiedin vivo since the production of these antibodies requires adysfunctional immune system comprising both B and T cells, and a failureto select and destroy immune cells that recognise self. However, invitro assays for T cell function (for example, a T cell proliferationassay) are appropriate screening tools to identify compounds that mayhave the ability to modulate the immune response in the complexsituation of autoimmune disease. Thus, a T cell proliferation assay isable to provide a measure of the effect of a compound on antinuclearantibodies.

Preparation of Peripheral Blood Mononuclear Cells

Two separate assays were conducted, Examples 3 and 4. Buffy coat bloodfrom three or four healthy donors (Example 3 and 4, respectively) wasobtained from Scottish National Blood Transfusion Services and separatedby centrifugation in BD Vacutainer™ CPT cell preparation tubes (4 mLdraw capacity, with sodium citrate, REF362781) at 300 g for 30 minutes,room temperature (RT). Peripheral blood mononuclear cell (PBMC) layerwas recovered by pipetting and pooled in 50 mL centrifuge tubes, thenwashed twice in 3 volumes Hank's buffered salt solution (w/o CaCl₂ andMgCl₂, Gibco #14175-053) by centrifuging at 300 g for 15 minutes (RT).PBMCs were resuspended in RPMI 1640 culture medium supplemented with 10%fetal calf serum and the number of viable cells was determined by TrypanBlue exclusion.

Cell Stimulation and Treatment

PBMCs were seeded in 96 well plates at 1×10⁵ cells/well, in 50 μL perwell culture medium. As assay control, CCRF-CEM leukaemia and LP-1multiple myeloma cells were seeded at 4000 and 5000 cells/well in 100μL/well RPMI 1640 tissue culture medium/10% fetal calf serum (FCS),respectively, and grown without stimulation or compound treatment forthe duration of the experiment. All stimulants and compounds were madeup to 4× final concentration in tissue culture medium and 25 μL of eachdrug and stimulant was then added to wells, for a final volume of 100μL/well. For unstimulated controls, 25 μL tissue culture medium wasadded to wells instead of stimulant and for untreated cells, 25 μL/welltissue culture medium containing equal volume DMSO (0.1% final) wasadded. Three adjacent wells were treated for each stimulation/treatmentcondition and the mean value calculated for analysis.

For Example 3, cells were allowed to settle for 2 hours beforestimulating with either 50 μg/mL PHA (Phytohemagglutinin PHA-P, SigmaL9132) or 50/250 ng/mL PMA/I (Phorbol 12-myristate 13-acetate/Ionomycin,Sigma P8139/10634). In addition, PBMCs were stimulated with ConA(Concanavalin A, Sigma C5275) at concentrations ranging from 0.5 μg/mLto 100 μg/ml to identify its stimulatory effect and optimalconcentration. For Example 4, PBMCs were stimulated immediately afterseeding, with either 50 μg/mL as before or 10 μg/mL ConA.

For Example 3, stimulated and unstimulated PBMCs were treated withcompounds [1]-[10] at IC₅₀, 2×IC₅₀, 3×IC₅₀ and 4×IC₅₀ concentrations.Compounds were added at two hours before stimulation (time of seeding),at the time of stimulation and two hours after stimulation and cellsthen incubated at 37° C., 5% CO₂, for 50, 48 and 46 hours with compound,respectively, or until 48 hours after stimulation. For Example 4,stimulated PBMCs were treated with the above compounds, plus compounds[11] and [12], with all compounds tested at 0.5×IC₅₀ and 0.25×IC₅₀ inaddition to the previous concentrations. Compounds were addedimmediately after stimulation and cells incubated for 48 and 72 hours.IC₅₀s were based on the average 72 hour IC₅₀s from in-house cytotoxicityassays on tumour cell panels (Table 16), except for compounds [1] and[3] for which IC₅₀s were based on published IC₅₀s previously usedin-house, as no in-house IC₅₀ data was available.

BrdU ELISA and Alamar Blue Assay

Proliferation activity of PBMCs after stimulation and compound treatmentwas determined by measuring BrdU incorporation, using the CellProliferation ELISA, BrdU (calorimetric) kit (Roche #1 647 229). Thiskit is a non-radioactive replacement method for ³H thymidineincorporation. Cells were labelled with BrdU for two hours prior toharvesting. Plates were then centrifuged for 10 minutes at 300 g (RT),supernatant removed from wells by pipetting and cells dried for 1 hourat 60° C. FixDenat solution was added to plates (100 μL/well) andincubated for 30 minutes, then flicked to remove and anti-BrdU-PODsolution added to plates (100 μL/well) and incubated for 90 minutes.After 3 washes with wash solution (200 μL/well), plates were incubatedwith substrate solution (100 μL/well) for 30 minutes, then 1M H₂SO₄ wasadded (25 μL/well) and optical density read at 450 nM, on an AscentFluoroskan plate reader.

Viability of PBMCs was assessed using Alamar Blue reagent (Biosource #DAL1100). Fresh 20% Alamar Blue was made up in culture medium and 100 μLadded to each well for a 10% final concentration. Plates were incubatedwith Alamar Blue at 37°, 5% CO₂, for 3 hours prior to harvesting time(48 or 72 hours) and fluorescence then read using excitation wavelength535 nM and emission wavelength 595 nM, on a Tecan Ultra plate reader.

Example 3 Results

Stimulation with PHA increased BrdU incorporation approximately 7-foldcompared to unstimulated cells. Treatment with DMSO vehicle did notaffect either BrdU incorporation or viability of PHA stimulated, orunstimulated cells (FIG. 4). As shown in FIG. 5, treatment of PHAstimulated cells with all of the compounds tested reduced BrdUincorporation, in many cases down to the level seen in unstimulatedcells. Reduction in BrdU incorporation is evident at concentrations aslow as IC₅₀ and with the exceptions of compounds [9] and [10], thequantity of BrdU incorporation still remaining does not correlate withcompound concentration, together suggesting that the compounds havereached their maximum proliferation inhibitory effect at IC₅₀. Forcompounds [9] and [10] the effect is clearly concentration dependent,with 3-4 times more BrdU incorporation at IC₅₀ dropping to unstimulatedlevels at the higher concentrations.

For most compounds, the time of compound treatment relative tostimulation time did not have a great effect on the level of BrdUincorporation, most likely because the stimulation had not yet takeneffect in those first four hours after PBMC seeding. The exceptions tothis are compounds [2], [3] and [4] where, surprisingly, there is lessreduction in BrdU incorporation with compound pre-treatment (−2 h) thanwhen compound treatment was started at the same time, or afterstimulation (FIG. 5).

Similar to PHA treatment, PMA/I stimulation results in around seven foldincrease in BrdU incorporation over unstimulated cells, when in theabsence of compound (FIG. 5). Treatment with all compounds other thancompounds [4], [9] and [10] reduced BrdU incorporation even more than inPHA stimulated cells and generally to levels lower than those seen inunstimulated cells, at all concentrations. PBMCs stimulated with PMA/Iand treated with DMSO only showed very low viability in Alamar Blueassay, while BrdU incorporation was increased (FIG. 4), as mentionedabove. Similarly, BrdU incorporation still remaining after treatment ofPMA/I stimulated cells with compounds [4], [9] and [10] is higher thanin PHA stimulated cells, despite negative Alamar Blue values.

Titration of Concanavalin A shows that ConA stimulates PBMCs in aconcentration dependent manner, reaching maximum effect at 10 μg/mL.BrdU incorporation is then reduced to unstimulated levels (higherconcentrations), consistent with viability assessment and microscopeobservations indicating that PBMCs are not viable with the higher ConAconcentrations tested. When compared with PHA and PMA/I, stimulationwith 10 μg/ml ConA increases BrdU incorporation to a comparable level,or by about 6-fold over unstimulated PBMCs, without reducing viability(FIG. 7).

Example 4 Results

Example 3 concluded that all the compounds tested affect theproliferation activity of stimulated PBMC cells at concentrations as lowas IC₅₀. A subsequent experiment (Example 4) was carried out to studythe concentration dependence of this effect at drug concentrations lowerthan IC₅₀. Treatments were repeated as before, with the addition oftreatments at lower concentrations, 0.5× and 0.25×IC₅₀, and DMSOcontrol, for all compounds previously tested plus compounds [11] and[12]. Since the time of compound treatment relative to stimulation didnot appear to be central for inhibition of proliferation activity, PBMCswere stimulated and compound treated at the same time, immediately afterseeding. Cells were stimulated with either PHA as before or with 10μg/mL ConA.

PHA stimulation increased BrdU incorporation of DMSO treated cells byover 20-fold and ConA stimulation by over 10-fold, compared withunstimulated cells, at 48 hours, with further increase at 72 hours, andDMSO vehicle had no effect on either BrdU incorporation or viability(FIG. 8). As shown in FIG. 9, all compounds reduced BrdU incorporationin a concentration dependent manner, most reaching maximum effect atIC₅₀ or 2×IC₅₀ concentrations, when stimulated with PHA. This confirmsthe previous observation that concentration dependence was not detectedat concentrations above IC₅₀, since maximum effect had already beenreached. Overall, the same effect was seen for PBMCs stimulated withConA, although maximum effect was reached at slightly lowerconcentrations. This may be a result of the fact that BrdU signals dropto background levels at maximum effect and, as signals are generallyonly half as high for ConA stimulated cells as for PHA stimulated cells,assay sensitivity may be slightly less for the former.

As before, compounds [4], [9] and [10] behave slightly differently fromthe rest of the compounds. Consistent with the previous experiment,concentration dependency was clearly seen for compounds [9] and [10] atconcentrations above IC₅₀, which then extended to the lowerconcentrations. At 4×IC₅₀, BrdU incorporation was still decreasing andsignals were approaching background levels for ConA stimulated cells,but were slightly higher in PHA stimulated cells. Without wishing to bebound by theory, this suggests that proliferation activity will becompletely inhibited as with the other compounds, but higherconcentrations are needed to achieve this.

For cells treated with compound [4], BrdU signals do not reduce tobackground levels with either stimulation, even at 4×IC₅₀. Furthermore,little correlation was seen between BrdU incorporation and compoundconcentration at the higher concentrations, consistent with our previousresults. Without wishing to be bound by theory, this suggests thateffect of compound [4] on proliferation activity is maximised at around2×IC₅₀ as for most of the other compounds, but the degree of inhibitionachieved is lower.

Viability assessment by Alamar Blue assay showed that viability of PBMCsis reduced slightly with stimulation, but is then generally comparablebetween compound-treated, DMSO-treated and untreated PBMCs. Furthermore,no reduction in Alamar Blue counts is associated with increasingcompound concentrations, confirming that the decreasing BrdUincorporation is not caused by cytotoxicity effects of the compounds andis purely representative of inhibition of proliferation activity.

By way of summary, experiments have shown that treatment with any of aset of transcriptional inhibitors alters the effect of PHA or ConAstimulation on T lymphocytes, resulting in a complete inhibition ofproliferation activity in a concentration dependent manner. Overall, thesame trend is seen for PHA and ConA stimulated cells, although completeinhibition of proliferation appears to be reached at lower drugconcentrations in ConA stimulated cells than PHA stimulated cells. Thismay be an artefact of the lesser total stimulation achieved by ConA thanPHA. Most compounds follow the same pattern where some inhibition ofproliferation activity is first seen with concentrations as low as0.25×IC₅₀ or 0.5×IC₅₀, then reaching complete inhibition at around2×IC₅₀, with some differences as to how rapid the drop in proliferationactivity is with increasing compound concentrations. A noteworthyexample is compound [7], which reaches complete inhibition atconcentrations as low as 0.25×IC₅₀. However, the comparison betweenspecific concentrations of different compounds must be approached withcaution, since IC₅₀ values are based on averages from different cellpanels.

Exceptions to the described pattern are seen for compounds [9] and [10],which are needed at higher concentrations than the other compounds tohave the same effect, and for compound [4], which follows the sameconcentration dependence as the others but fails to achieve completeinhibition of proliferation activity at the higher concentrations.

Experiments have shown that PBMC viability is reduced slightly withstimulation but no significant further reduction is associated withcompound treatment. More importantly, viability is clearly not dependenton compound concentration and the measured reduction in BrdUincorporation therefore represents true inhibition of proliferationactivity and not cytotoxicity of the compounds.

Various modifications and variations of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention which are obvious to those skilledin the relevant fields are intended to be covered by the presentinvention.

REFERENCES

-   Balomenos D. et al. The cell cycle inhibitor p21 controls T-cell    proliferation and sex-linked lupus development. Nat. Med, 6:171-176,    2000.-   Corna D et al. Mycophenolate mofetil limits renal damage and    prolongs life in murine lupus disease. Kidney Int, 51:1583-1589,    1997.-   Foster M H. et al. Relevance of systemic lupus erythematosus    nephritis animal models to human disease. Semin Nephrol, 19:12-24,    1999.-   Gelfand M C et al. Therapeutic studies in NZB/W mice. I. Synergy of    azathioprine, cyclophosphamide and methylprednisolone in    combination. Arthritis Rheum, 15: 239-246, 1972.-   Kewalramani R. et al. Immunopathogenesis of lupus and lupus    nephritis:recent insights. Curr Opin Nephrol Hypertens, 11:273-277,    2002.-   Von Mulen C. A. et al. Autoantibodies in the Diagnosis of Systemic    Rheumatic Diseases. Semin Arthritis Rheum 1995; 24:323-58.-   Peutz-Kootstra C. J. et al. Lupus nephritis:lessons from    experimental animal models. J Lab Clin Med, 137:244-259, 2001.-   Rekvig O P et al. Anti-double-stranded DNA antibodies, nucleosomes,    and systemic lupus erythematosus. Arthritis & Rheumatism 48:    300-312, 2003.-   Reichlin M. et al. Antinuclear Antibodies: An Overview, Dubois'    Lupups Erythematosus 5^(th) Edn, eds Wallace D. J. and Hahn B. H.,    Williams, Wilkins and Baltimore, 1997, p397-405.-   Shankland S. J. et al. Cell cycle regulatory proteins in renal    disease: role in hypertrophy, proliferation, and apoptosis. Am J    Physiol Renal Physiol, 278:F515-F529, 2000.-   Van Venrooij W. J. et al. Manual of Biological Markers of Disease,    Section B: Autoantigens. Kluwer Academic Publishing, 1994.-   Xu L. et al. Human lupus T cells resist inactivation and escape    death by upregulating COX-2. Nature Medicine, 10:411-415, 2004.-   Zoja C. et al. Renal expression of monocyte chemoattractant    protein-1 in lupus autoimmune mice. J Am Soc Nephrol 8:720-729,    1997.-   Zoja C. et al. Bindarit retards renal disease and prolongs survival    in murine lupus autoimmune disease. Kidney Int, 53:726-734, 1998.-   Zoja C. et al. Mycophenolate mofetil combined with a    cyclooxygenase-2 inhibitor ameliorates murine lupus nephritis.    Kidney Int, 60:653-663, 2001.

TABLE 1 Body Weight 8 2 3 4 5 6 7 months Vehicle 27.69 ± 0.61 27.85 ±0.59 28.31 ± 0.56 28.08 ± 0.79 29.91 ± 0.65 31.60 ± 0.75 31.75 ± 1.11CYC202 27.85 ± 0.72 28.00 ± 0.68  28.54 ± 0 0.45 28.92 ± 0.35 31.58 ±0.43 30.82 ± 0.35 31.60 ± 0.52 (200 mg/kg) Preventive study CYC202 27.93± 0.62 27.86 ± 0.52 28.07 ± 0.57 28.64 ± 0.57 30.23 ± 0.67 31.50 ± 0.8730.78 ± 0.91 (100 mg/kg) Preventive study CYC202 26.71 ± 0.74 28.14 ±0.69 29.43 ± 1.01 29.71 ± 0.82 31.45 ± 0.96 30.80 ± 1.69 29.80 ± 1.46(100 mg/kg) Therapeutic study Values are expressed as mean ± SE

TABLE 2 Food Intake 5 2 2.5 3 3.5 4 4.5 months Vehicle 3.77 ± 0.28 4.31± 0.17 4.15 ± 0.22 3.62 ± 035 3.54 ± 0.35 3.31 ± 0.35 3.50 ± 0.15 CYC2023.54 ± 0.29 4.08 ± 0.08 4.00 ± 0.11 4.08 ± 0.21 4.00 ± 0.16 4.00 ± 0.183.92 ± 0.29 (200 mg/kg) Preventive study CYC202 3.69 ± 0.33 3.77 ± 0.233.92 ± 0.18 4.08 ± 0.18 3.92 ± 0.26 3.85 ± 0.30 3.85 ± 0.22 (100 mg/kg)Preventive study Values are expressed as mean ± SE

TABLE 3 Water intake 5 2 2.5 3 3.5 4 4.5 months Vehicle 4.62 ± 0.33 5.00± 0.30 5.00 ± 0.30 4.54 ± 0.39 5.15 ± 0.15 5.00 ± 0.23 5.08 ± 0.23CYC202 5.31 ± 0.26 4.77 ± 0.20 4.92 ± 0.18 5.08 ± 0.21 4.85 ± 0.27 4.38± 0.43 4.23 ± 0.28 (200 mg/kg) Preventive study CYC202 6.64 ± 0.29 5.14± 0.40 4.21 ± 0.28 5.00 ± 0.28 5.57 ± 0.20 4.43 ± 0.20 3.93 ± 0.22 (100mg/kg Preventive Study Values are expressed as mean ± SE

TABLE 4 Survival % 8 3 4 5 5.5 6 6.5 7 7.5 months Vehicle 100 100 92 8585 62 38 31 31 CYC202 100 100 100 92 92 92 85 77 77 (200 mg/kg)Preventive study CYC202 100 100 100 100 93 71 71 71 71 (100 mg/kg)Preventive study CYC202 100 100 100 93 71 64 36 36 36 (100 mg/kg)Therapeutic study

TABLE 5 Cumulative precentage of proteinuric mice 3 4 5 5.5 6 6.5 7 7.5Vehicle 0 0 7.7 46.0  69.0 77.0 77.0 85 CYC2002 0 0 0 23.1  23.1* 23.1**23.1** 23.1** (200 mg/kg) CYC202 0 0 0  0** 21.4* 21.4** 42.9 42.9* (100mg/kg) CYC202 0 0 7.1 21.4  28.5 35.7 50.0 57 (100 mg/kg) *P < 0.05, **P< 0.01 vs vehicle

TABLE 6 Renal function assessed by serum BUN 8 Months Vehicle 50%(43-169 mg/dl) CYC202 30% (200 mg/kg) (36-88 mg/dl) Preventive studyCYC202 33% (100 mg/kg) (31-43 mg/dl) Preventive study CYC202 40% (100mg/kg) (30-111 mg/dl) Therapeutic study

TABLE 7 Serum anti-DNA antibodies (U/ml) Vehicle 5 months 181.30 ± 74.158 months 367.29 ± 149.02 CYC202 5 months  24.20 ± 6.52 (200 mg/kg) 8months  73.39 ± 21.13* Prventive study CYC202 5 months  4.04 ± 0.76**(100 mg/kg) 8 months  76.55 ± 21.40* Preventive study CYC202 8 months153.22 ± 90.73 (100 mg/kg) Therapeutic study Control (CD-1  6.43 ± 1.34mice) Values are expressed as mean ± SE *P < 0.05, **P < 0.01 vs vehicleat corresponding time.

TABLE 8 Serum Transaminase (IU/L) AST ALT (IU/L) (IU/L) CYC202 62.89 ±4.70 36.44 ± 4.04 (200 mg · kg) Preventive study CYC202 74.40 ± 1.7236.00 ± 2.45 (100 mg/kg) Therapeutic study Control (CD-1 78.00 ± 2.2836.40 ± 2.64 mice) Values are expressed as mean ± SE

TABLE 9 Renal Histology Intracapillary Extracapillary GlomeruliTubulointerstital hypercellularity Proliferation deposits Damage Vehicle2.00 ± 0.44 1.00 ± 0.38 1.14 ± 0.34 0.71 ± 0.23 CYC202 0.36 ± 0.15**°°0.18 ± 0.18 0.27 ± 0.19* 0.25 ± 0.16*° (200 mg/kg) Preventive studyCYC202 0.92 ± 0.23* 0.67 ± 0.26 0.92 ± 0.23 0.46 ± 0.17 (100 mg/kg)Preventive study CYC202 1.29 ± 0.18 0.57 ± 0.20 1.14 ± 0.26 0.61 ± 013(100 mg/kg) Therapeutic study Values are expressed as mean ± SE. *P <0.05, **P < 0.05, °°P < 0.01 vs CYC202 (100 mg/kg) from 5 months

TABLE 10 F4/80 positive monocytes/macrophages in the renal interstitium8 months Vehicle 29.68 ± 5.97# CYC202 (200 mg/kg)  8.56 ± 1.94°*#Preventive study CYC202 (100 mg/kg() 18.26 ± 4.89# Preventive studyCYC202 (100 mg/kg) 21.87 ± 5.13# Therapeutic study Control  0.25 ± 0.25

TABLE 11 Survival % 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 m(pre-treatment) Vehicle 100 100 100 90 90 90 60 30 20 10 10 10 10 10 0CYC202 100 100 93 87 87 80 80 67 53 40 34 20 13 13 13 (200 mg/kg) MPS100 100 100 92 75 67 67 67 42 42 33 33 33 33 33 (1.5 mg/kg) CYC202 + MPS100 100 100 94 94 94 94 94 81 75 69 62 62 62 62 Treatments started at 5months of age. CYC202 + MPS significantly (P < 0.0001) prolonged life inrespect to vehicle.

TABLE 12 Cumulative percentage of mice with proteinuria >4 mg/day 5 5.56 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 m (pre-treatment) Vehicle 010 20 30 40 90 90 90 90 90 90 90 90 90 90 CYC202 0 0 6.7 26.7 26.7  46.6* 66.6 66.6 73.3 73.3 73.3 73.3 73.3 73.3 73.3 (200 mg/kg) MPS 0 016.7 33.3 33.3 50 75 75 75 75 75 75 75 75 75 (1.5 mg/kg) CYC202 + MPS 06.2 6.2 6.2 6.2*  25** 37.5* 43.8* 43.8* 43.8* 43.8* 50 56.2 56.2 56.2Each point reflects the current level of proteinuria in surviving miceas wellas the last measurement in deceased mice. 0.05, **p < 0.01vsvehicle.

TABLE 13 Renal Function - Cumulative % mice with BUN >30 mg/dl 12 5 6 78 9 10 11 months Vehicle 0 10% 40% 80% 90% 90% 90% 100% CYC202 0% 7% 15%43% 57% 64% 64% 64% (200 mg/kg) MPS 0% 0% 17% 42% 50% 58% 58% 67% (1.5mg/kg) CYC202 + 0% 0% 0% 27% 40% 40% 47% 53% MPS BUN levels >30 mg/dlwere considered abnormal (normal range: 14-29 mg/dl)

TABLE 14 Renal Histology GLOMERULI Intracapillary extracapillary immuneTUBULO-INTERSTITIAL hypercellularity proliferation deposits DAMAGEVehicle 1.60 ± 0.24 1.00 ± 0.00 2.20 ± 0.37 1.70 ± 0.09 CYC202 1.14 ±0.26 0.86 ± 0.14 1.43 ± 0.30 1.29 ± 0.23 (200 mg/kg) MPS 1.40 ± 0.400.80 ± 0.20 1.80 ± 0.20 1.20 ± 0.14 (1.5 mg/kg) CYC202 + MPS 0.70 ± 0.300.30 ± 0.15* 0.90 ± 0.23*° 0.45 ± 0.21** Values are expressed as meanscore ± SE. *p < 0.05, **p < 0.01 vs vehicle; °p < 0.05 vs MPS.

TABLE 15 F4/80 monocytes/macrophages in the renal interstitium(Cells/HPF) Vehicle 61.5 ± 4.6 CYC202 (200 mg/kg) 41.4 ± 8.6 MPS (1.5mg/kg) 57.2 ± 10.5 CYC202 + MPS 21.8 ± 4.8**° Values are expressed asmean ± SE. HPF = high power field. **p < 0.01 vs vehicle, °p < 0.05 vs<PS Control CD-1 mice range: 0-4 cells/HPF

TABLE 16 Average 72 hours IC₅₀ values (μM) for in-house cell panel androunded IC₅₀ value used, for ease of calculation IC₅₀ Number of IC₅₀Compound Average Cell lines Used 1 27*  1* 25* 2 16.45 62 16 3 56*  1*50* 4  1.04 47  1 5  1.43 13  1.5 6 10.4 13 10 7  5.31 18  5 8  0.876 35 1 9  0.250  6  0.25 10  0.753 11  1 11  0.291  7  0.5 12  0.353  7  0.5*No in-house cytotoxicity data available, IC₅₀s published for HCT116cells in Raynaud et.al. Clin Cancer Res 11 (13): 4875-87, 2005.

TABLE 17 Average IC₅₀ values (μM) for compounds against CDK/cyclin andAurora enzymes from in-house kinase assays Compound CDK1B CDK2A CDK2ECDK4D1 CDK6D3 CDK7H CDK9T1 AurA AurB 1 >100 8.84 0.946 45.7 n/a 0.6032.03 n/a n/a 2 17.5 2.80 0.208 27.5 55.5 0.438 1.04 >100 n/a 3 >100.00083.08 7.67 >100 n/a >20 2.72 n/a n/a 4 7.46 1.05 0.505 2.82 1.04 8.571.88 0.038 0.030 5 15.9 1.24 0.931 2.75 1.94 27.0 2.71 0.051 0.023 67.60 0.373 0.126 19.3 9.19 1.33 0.338 >10 >10 7 4.95 0.223 0.071 7.636.27 0.983 0.195 >10 >10 8 1.99 2.00 0.354 0.089 0.168 0.053 0.034 0.6520.399 9 2.00 1.67 0.204 0.118 0.110 0.147 0.097 0.094 0.021 10 1.490.115 0.013 5.10 6.40 0.455 0.081 >10 >100 11 0.667 0.304 0.054 0.066n/a 0.266 0.029 0.028 0.008 12 1.03 0.076 0.025 1.02 n/a 2.38 0.2150.161 0.025

1. (canceled)
 2. The method according to claim 12 wherein the disease isan autoimmune rheumatic disease or organ specific autoimmunity.
 3. Themethod according to claim 2 wherein the autoimmune rheumatic disease isselected from drug-induced lupus, systemic lupus erythematosis (SLE),and rheumatoid arthritis.
 4. The method according to claim 3 wherein theautoimmune rheumatic disease is systemic lupus erythematosis (SLE). 5.The method according to claim 12 wherein the antinuclear antibodies areanti-DNA antibodies.
 6. The method according to claim 12 wherein theinhibitor of CDK2 and/or CDK7 and/or CDK9 is in an amount sufficient todown-regulate the expression of Mcl-1.
 7. The method according to claim12 wherein the inhibitor of CDK2 and/or CDK7 and/or CDK9 is in an amountsufficient to delay the onset of proteinuria and renal functionimpairment.
 8. The method according to claim 12 wherein the inhibitor ofCDK2 and/or CDK7 and/or CDK9 is in an amount sufficient to reduceglomerular and tubointerstitial changes.
 9. The method according toclaim 12 wherein the inhibitor of CDK2 and/or CDK7 and/or CDK9 is in anamount sufficient to reduce the interstitial accumulation of mononuclearcells.
 10. The method according to claim 12 wherein the inhibitor ofCDK2 and/or CDK7 and/or CDK9 is in an amount sufficient to inhibitT-cell proliferation (induced by PMA and ConA).
 11. The method accordingto claim 12 wherein the the inhibitor is administered in combinationtherapy with methylprednisolone.
 12. A method of treating a diseaseassociated with antinuclear antibodies in a subject, said methodcomprising administering to the subject an inhibitor of CDK2 and/or CDK7and/or CDK9, or a pharmaceutically acceptable salt thereof, in an amountsufficient to down-regulate the level of antinuclear antibodies.
 13. Amethod of treating an autoimmune disease in a subject by down-regulatingthe level of antinuclear antibodies in said subject, said methodcomprising administering an inhibitor of CDK2 and/or CDK7 and/or CDK9,or a pharmaceutically acceptable salt thereof, in an amount sufficientto down-regulate the level of antinuclear antibodies.
 14. A method oftreating a disease associated with antinuclear antibodies in a subjectby down-regulating the level of antinuclear antibodies, said methodcomprising administering an inhibitor of CDK2 and/or CDK7 and/or CDK9,or a pharmaceutically acceptable salt thereof, such that said disease istreated.
 15. A method of down-regulating the level of antinuclearantibodies in a subject, said method comprising administering aninhibitor of CDK2 and/or CDK7 and/or CDK9, or a pharmaceuticallyacceptable salt thereof, to said subject in an amount sufficient todown-regulate the level of antinuclear antibodies.
 16. A methodaccording to any one of claims 12 to 15 wherein the disease is anautoimmune rheumatic disease or organ specific autoimmunity.
 17. Amethod according to claim 16 wherein the autoimmune rheumatic disease isselected from drug induced lupus, systemic lupus erythematosis (SLE),and rheumatoid arthritis.
 18. A method according claim 17 wherein theautoimmune rheumatic disease is systemic lupus erythematosis (SLE). 19.A method according to any one of claims 12 to 15 wherein the antinuclearantibodies are anti-DNA antibodies.
 20. A method according to any one ofclaims 12 to 15 wherein the inhibitor of CDK2 and/or CDK7 and/or CDK9 isin an amount sufficient to down-regulate the expression of Mcl-1.
 21. Amethod according to any one of claims 12 to 15 wherein the inhibitor ofCDK2 and/or CDK7 and/or CDK9 is in an amount sufficient to delay theonset of proteinuria and renal function impairment.
 22. A methodaccording to any one of claims 12 to 15 wherein the inhibitor of CDK2and/or CDK7 and/or CDK9 is in an amount sufficient to reduce glomerularand tubulointerstitial changes.
 23. A method according to any one ofclaims 12 to 15 wherein the inhibitor of CDK2 and/or CDK7 and/or CDK9 isin an amount sufficient to reduce the interstitial accumulation ofmononuclear cells.
 24. A method according to any one of claims 12 to 15wherein the inhibitor of CDK2 and/or CDK7 and/or CDK9 is in an amountsufficient to inhibit T-cell proliferation induced by PMA and ConA. 25.A method according to any one of claims 12 to 15 which further comprisesadministrating methylprednisolone to the subject.
 26. A method accordingto claim 25 wherein the methylprenisolone and the inhibitor of CDK2and/or CDK7 and/or CDK9, or pharmaceutically acceptable salt thereof,are administered simultaneously, sequentially or separately.
 27. Amethod of down-regulating the level of antinuclear antibodies in a cell,said method comprising contacting said cell with an inhibitor of CDK2and/or CDK7 and/or CDK9, or a pharmaceutically acceptable salt thereof,in an amount sufficient to down-regulate the level of antinuclearantibodies in said cell.
 28. A pharmaceutical composition for treating adisease associated with antinuclear antibodies, said compositioncomprising an inhibitor of CDK2 and/or CDK7 and/or CDK9, in an amountsufficient to down-regulate the level of antinuclear antibodies, admixedwith a pharmaceutically acceptable diluent, excipient or carrier.
 29. Acombination comprising an inhibitor of CDK2 and/or CDK7 and/or CDK9, ora pharmaceutically acceptable salt thereof, and methylprednisolone. 30.A pharmaceutical composition comprising a combination according to claim29 and a pharmaceutically acceptable carrier, diluent or excipient. 31.A pharmaceutical product comprising an inhibitor of CDK2 and/or CDK7and/or CDK9, or a pharmaceutically acceptable salt thereof, andmethylprednisolone as a combined preparation for simultaneous,sequential or separate use in therapy.
 32. A pharmaceutical productaccording to claim 31 wherein the inhibitor of CDK2 and/or CDK7 and/orCDK9, or pharmaceutically acceptable salt thereof, andmethylprednisolone are administered simultaneously.
 33. A pharmaceuticalproduct according to claim 31 which further comprises a pharmaceuticallyacceptable carrier, diluent or excipient.
 34. A pharmaceutical productaccording to claim 31 for use in the treatment of a disease associatedwith antinuclear antibodies.
 35. A pharmaceutical product according toclaim 34 wherein the disease is an autoimmune rheumatic disease or organspecific autoimmunity.
 36. A pharmaceutical product according to claim35 wherein the autoimmune rheumatic disease is selected from druginduced lupus, systemic lupus erythematosis (SLE), and rheumatoidarthritis.
 37. A pharmaceutical product according to claim 36 whereinthe autoimmune rheumatic disease is systemic lupus erythematosis (SLE).38. A pharmaceutical composition comprising: (i) an inhibitor of CDK2and/or CDK7 and/or CDK9, or a pharmaceutically acceptable salt thereof;and (ii) methylprednisolone; admixed with a pharmaceutically acceptablediluent, excipient or carrier.
 39. A pharmaceutical compositionaccording to claim 28, wherein the inhibitor of CDK2 and/or CDK7 and/orCDK9 is a purine derivative or a pyrimidine derivative.
 40. Apharmaceutical composition according to claim 28, wherein the inhibitorof CDK2 and/or CDK7 and/or CDK9 is selected from the following:

and pharmaceutically acceptable salts thereof.
 41. A pharmaceuticalcomposition according to claim 28, wherein the inhibitor of CDK2 and/orCDK7 and/or CDK9 is selected from roscovitine, olomoucine and purvalanolA, and pharmaceutically acceptable salts thereof.
 42. A pharmaceuticalcomposition according to claim 28, wherein the inhibitor of CDK2 and/orCDK7 and/or CDK9 is roscovitine, or a pharmaceutically acceptable saltthereof. 43-50. (canceled)
 51. A method according to claim 53 whereinthe antinuclear antibodies are anti-DNA antibodies.
 52. A methodaccording to claim 53 wherein the level of antinuclear antibodies isreduced.
 53. A method of treating a disease associated with antinuclearantibodies in a subject, said method comprising administering to thesubject a therapeutically acceptable amount of: (i) an inhibitor of CDK2and/or CDK7 and/or CDK9, or a pharmaceutically acceptable salt thereof;and (ii) methylprednisolone.
 54. A method according to claim 53 whereinan inhibitor of CDK2 and/or CDK7 and/or CDK9, or pharmaceuticallyacceptable salt thereof, and methylprednisolone are admixed with apharmaceutically acceptable diluent, excipient or carrier.
 55. A methodaccording to claim 53 wherein the inhibitor of CDK2 and/or CDK7 and/orCDK9, or pharmaceutically acceptable salt thereof, andmethylprednisolone are administered simultaneously, separately orsequentially.
 56. A method according to claim 55 which comprisesadministering an inhibitor of CDK2 and/or CDK7 and/or CDK9, or apharmaceutically acceptable salt thereof, to a subject prior tosequentially or separately administering methylprednisolone to saidsubject.
 57. A method according to claim 55 which comprisesadministering methylprednisolone to a subject prior to sequentially orseparately administering an inhibitor of CDK2 and/or CDK7 and/or CDK9,or a pharmaceutically acceptable salt thereof.
 58. A method according toclaim 53 wherein the inhibitor of CDK2 and/or CDK7 and/or CDK9, orpharmaceutically acceptable salt thereof, and methylprednisolone areeach administered in a therapeutically effective amount with respect tothe individual components.
 59. A method according to claim 53 whereinthe inhibitor of CDK2 and/or CDK7 and/or CDK9, or pharmaceuticallyacceptable salt thereof, and methylprednisolone are each administered ina subtherapeutic amount with respect to the individual components.
 60. Amethod according to claim 53 wherein the disease is an autoimmunerheumatic disease or organ specific autoimmunity.
 61. A method to claim60 wherein the autoimmune rheumatic disease is selected from druginduced lupus, systemic lupus erythematosis (SLE), and rheumatoidarthritis.
 62. A method according to claim 61 wherein the autoimmunerheumatic disease is systemic lupus erythematosis (SLE).
 63. A methodaccording claim 12 or 53, wherein the inhibitor of CDK2 and/or CDK7and/or CDK9 is a purine derivative or a pyrimidine derivative.
 64. Amethod according claim 12 or 53, wherein the inhibitor of CDK2 and/orCDK7 and/or CDK9 is selected from the following:

and pharmaceutically acceptable salts thereof.
 65. A method accordingclaim 12 or 53, the inhibitor of CDK2 and/or CDK7 and/or CDK9 isselected from roscovitine, olomoucine and purvalanol A.
 66. A methodaccording claim 12 or 53, the inhibitor of CDK2 and/or CDK7 and/or CDK9is roscovitine, or a pharmaceutically acceptable salt thereof. 67.(canceled)